Methods of using [3.2.0] heterocyclic compounds and analogs thereof

ABSTRACT

Disclosed are methods of treating cancer, inflammatory conditions, and/or infectious disease in an animal comprising: administering to the animal, a therapeutically effective amount of a heterocyclic compound. The animal is a mammal, preferably a human or a rodent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 60/480,270, filed on Jun. 20, 2003, entitledUSE OF SALINOSPORAMIDE A TO TREAT LeTx INTOXICATION AND B. ANTHRACISINFECTION, and to U.S. Provisional Application No. 60/566,952, filed onApr. 30, 2004, entitled METHODS OF USING (3.2.0) HETEROCYCLIC COMPOUNDSAND ANALOGS THEREOF; the disclosures of both of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to certain compounds and to methods forthe preparation and the use of certain compounds in the fields ofchemistry and medicine. Embodiments of the invention disclosed hereinrelate to methods of using heterocyclic compounds. In some embodiments,the compounds are used as proteasome inhibitors. In other embodiments,the compounds are used to treat inflammation, cancer, and infectiousdiseases.

2. Description of the Related Art

Cancer is a leading cause of death in the United States. Despitesignificant efforts to find new approaches for treating cancer, theprimary treatment options remain surgery, chemotherapy and radiationtherapy, either alone or in combination. Surgery and radiation therapy,however, are generally useful only for fairly defined types of cancer,and are of limited use for treating patients with disseminated disease.Chemotherapy is the method that is generally useful in treating patientswith metastatic cancer or diffuse cancers such as leukemias. Althoughchemotherapy can provide a therapeutic benefit, it often fails to resultin cure of the disease due to the patient's cancer cells becomingresistant to the chemotherapeutic agent. Due, in part, to the likelihoodof cancer cells becoming resistant to a chemotherapeutic agent, suchagents are commonly used in combination to treat patients.

Similarly, infectious diseases caused, for example, by bacteria, fungiand protozoa are becoming increasingly difficult to treat and cure. Forexample, more and more bacteria, fungi and protozoa are developingresistance to current antibiotics and chemotherapeutic agents. Examplesof such microbes include Bacillus, Leishmania, Plasmodium andTrypanosoma.

Furthermore, a growing number of diseases and medical conditions areclassified as inflammatory diseases. Such diseases include conditionssuch as asthma to cardiovascular diseases. These diseases continue toaffect larger and larger numbers of people worldwide despite newtherapies and medical advances.

Therefore, a need exists for additional chemotherapeutics,anti-microbial agents, and anti-inflammatory agents to treat cancer,inflammatory diseases and infectious disease. A continuing effort isbeing made by individual investigators, academia and companies toidentify new, potentially useful chemotherapeutic and anti-microbialagents.

Marine-derived natural products are a rich source of potential newanti-cancer agents and anti-microbial agents. The oceans are massivelycomplex and house a diverse assemblage of microbes that occur inenvironments of extreme variations in pressure, salinity, andtemperature. Marine microorganisms have therefore developed uniquemetabolic and physiological capabilities that not only ensure survivalin extreme and varied habitats, but also offer the potential to producemetabolites that would not be observed from terrestrial microorganisms(Okami, Y. 1993 J Mar Biotechnol 1:59). Representative structuralclasses of such metabolites include terpenes, peptides, polyketides, andcompounds with mixed biosynthetic origins. Many of these molecules havedemonstrable anti-tumor, anti-bacterial, anti-fungal, anti-inflammatoryor immunosuppressive activities (Bull, A. T. et al. 2000 Microbiol MolBiol Rev 64:573; Cragg, G. M. & D. J. Newman 2002 Trends Pharmacol Sci23:404; Kerr, R. G. & S. S. Kerr 1999 Exp Opin Ther Patents 9:1207;Moore, B. S 1999 Nat Prod Rep 16:653; Faulkner, D. J. 2001 Nat Prod Rep18:1; Mayer, A. M. & V. K. Lehmann 2001 Anticancer Res 21:2489),validating the utility of this source for isolating invaluabletherapeutic agents. Further, the isolation of novel anti-cancer andanti-microbial agents that represent alternative mechanistic classes tothose currently on the market will help to address resistance concerns,including any mechanism-based resistance that may have been engineeredinto pathogens for bioterrorism purposes.

SUMMARY OF THE INVENTION

The embodiments disclosed herein generally relate to chemical compounds,including heterocyclic compounds and analogs thereof. Some embodimentsare directed to the use of compounds as proteasome inhibitors.

In other embodiments, the compounds are used to treat neoplasticdiseases, for example, to inhibit the growth of tumors, cancers andother neoplastic tissues. The methods of treatment disclosed herein maybe employed with any patient suspected of carrying tumorous growths,cancers, or other neoplastic growths, either benign or malignant(“tumor” or “tumors” as used herein encompasses tumors, cancers,disseminated neoplastic cells and localized neoplastic growths).Examples of such growths include but are not limited to breast cancers;osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas;leukemias; sinus tumors; ovarian, uretal, bladder, prostate and othergenitourinary cancers; colon, esophageal and stomach cancers and othergastrointestinal cancers; lung cancers; lymphomas; myelomas; pancreaticcancers; liver cancers; kidney cancers; endocrine cancers; skin cancers;melanomas; angiomas; and brain or central nervous system (CNS) cancers.In general, the tumor or growth to be treated may be any tumor orcancer, primary or secondary. Certain embodiments relate to methods oftreating neoplastic diseases in animals. The method can include, forexample, administering an effective amount of a compound to a patient inneed thereof. Other embodiments relate to the use of compounds in themanufacture of a pharmaceutical or medicament for the treatment of aneoplastic disease. The compounds can be administered in combinationwith a chemotherapeutic agent.

In still other embodiments, the compounds are used to treat inflammatoryconditions. Certain embodiments relate to methods of treatinginflammatory conditions in animals. The method can include, for example,administering an effective amount of a compound to a patient in needthereof. Other embodiments relate to the use of compounds in themanufacture of a pharmaceutical or medicament for the treatment ofinflammation.

In certain embodiments, the compounds are used to treat infectiousdiseases. The infectious agent can be a microbe, for example, bacteria,fungi, protozoans, and microscopic algae, or viruses. Further, theinfectious agent can be B. anthracis (anthrax). In some embodiments theinfectious agent is a parasite. For example, the infectious agent can bePlasmodium, Leishmania, and Trypanosoma. Certain embodiments relate tomethods of treating infectious agents in animals. The method caninclude, for example, administering an effective amount of a compound toa patient in need thereof. Other embodiments relate to the use ofcompounds in the manufacture of a pharmaceutical or medicament for thetreatment of infectious agents.

Some embodiments relate to uses of a compound having the structure ofFormula I, and pharmaceutically acceptable salts and pro-drug estersthereof:

wherein the dashed lines represent a single or a double bond, wherein R1may be separately selected from the group consisting of a hydrogen, ahalogen, mono-substituted, poly-substituted or unsubstituted variants ofthe following residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, andhalogenated alkyl including polyhalogenated alkyl, where n is equal to 1or 2, and if n is equal to 2, then R₁ can be the same or different;

-   -   wherein R₂, may be selected from the group consisting of        hydrogen, a halogen, mono-substituted, poly-substituted or        unsubstituted variants of the following residues: saturated        C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,        acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy,        cycloalkyl, cycloalkenyl (including, for example,        cyclohexylcarbinol), alkoxy, cycloalkoxy, aryl, heteroaryl,        arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,        aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,        alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and        halogenated alkyl including polyhalogenated alkyl;    -   wherein R₃ may be selected from the group consisting of a        halogen, mono-substituted, poly-substituted or unsubstituted        variants of the following residues: saturated C₁-C₂₄ alkyl,        unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,        alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy        carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl,        aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl, hydroxy,        alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and        halogenated alkyl including polyhalogenated alkyl; and wherein        each of E₁, E₂, E₃ and E₄ is a substituted or unsubstituted        heteroatom; in the treatment of cancer, inflammation, and        infectious disease.

Other embodiments relate to methods of treating a neoplastic disease inan animal. The methods can include, for example, administering to theanimal, a therapeutically effective amount of a compound of a formulaselected from Formulae I-V, and pharmaceutically acceptable salts andpro-drug esters thereof.

Further embodiments relate to pharmaceutical compositions which includea compound of a formula selected from Formulae I-V. The pharmaceuticalcompositions can further include an anti-microbial agent.

Still further embodiments relate to methods of inhibiting the growth ofa cancer cell. The methods can include, for example, contacting a cancercell with a compound of a formula selected from Formulae I-V, andpharmaceutically acceptable salts and pro-drug esters thereof.

Other embodiments relate to methods of inhibiting proteasome activitythat include the step contacting a cell with a compound of a formulaselected from Formulae I-V, and pharmaceutically acceptable salts andpro-drug esters thereof.

Other embodiments relate to methods of inhibiting NF-κB activationincluding the step contacting a cell with a compound of a formulaselected from Formulae I-V, and pharmaceutically acceptable salts andpro-drug esters thereof.

Some embodiments relate to methods for treating an inflammatorycondition, including administering an effective amount of a compound ofa formula selected from Formulae I-V to a patient in need thereof.

Further embodiments relate to methods for treating a microbial illnessincluding administering an effective amount of a compound of a formulaselected from Formulae I-V to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form part ofthe specification, merely illustrate certain preferred embodiments ofthe present invention. Together with the remainder of the specification,they are meant to serve to explain preferred modes of making certaincompounds of the invention to those of skilled in the art. In thedrawings:

FIG. 1 shows the chemical structure of Salinosporamide A.

FIG. 2 shows the pan-tropical distribution of the Salinospora. “X”denotes Salinospora collection sites.

FIG. 3 shows colonies of Salinospora.

FIG. 4 shows the typical 16S rDNA sequence of the Salinospora. Barsrepresent characteristic signature nucleotides of the Salinospora thatseparate them from their nearest relatives.

FIG. 5 shows Omuralide, a degradation product of the microbialmetabolite Lactacystin. Also shown is a compound of Formula II-16, alsoreferred to as Salinosporamide A.

FIG. 6 illustrates lethal toxin-mediated macrophage cytotoxicity.NPI-0052 represents the compound of Formula II-16.

FIG. 7 depicts the 1H NMR spectrum of a compound having structureFormula II-20.

FIG. 8 depicts the 1H NMR spectrum of a compound having structureFormula II-24C.

FIG. 9 depicts the 1H NMR spectrum of a compound having structureFormula II-19.

FIG. 10 depicts the 1H NMR spectrum of a compound having structureFormula II-2.

FIG. 11 depicts the mass spectrum of a compound having structure FormulaII-2.

FIG. 12 depicts the 1H NMR spectrum of a compound having structureFormula II-3.

FIG. 13 depicts the mass spectrum of a compound having structure FormulaII-3.

FIG. 14 depicts the 1H NMR spectrum of a compound having structureFormula II-4.

FIG. 15 depicts the mass spectrum of a compound having structure FormulaII-4.

FIG. 16 depicts the 1H NMR spectrum of a compound having structureFormula II-5A.

FIG. 17 depicts the mass spectrum of a compound having structure FormulaII-5A.

FIG. 18 depicts the 1H NMR spectrum of a compound having structureFormula II-5B.

FIG. 19 depicts the mass spectrum of a compound having structure FormulaII-5B.

FIG. 20 depicts the 1H NMR spectrum of a compound having structureFormula IV-3C in DMSO-d₆.

FIG. 21 depicts the 1H NMR spectrum of a compound having structureFormula IV-3C in C₆D₆/DMSO-d₆.

FIG. 22 depicts the 1H NMR spectrum of a compound having structureFormula II-13C.

FIG. 23 depicts the 1H NMR spectrum of a compound having structureFormula II-8C.

FIG. 24 depicts the 1H NMR spectrum of a compound having structureFormula II-25.

FIG. 25 depicts the 1H NMR spectrum of a compound having structureFormula II-21.

FIG. 26 depicts the 1H NMR spectrum of a compound having structureFormula II-22.

FIG. 27 shows inhibition of the chymotrypsin-like activity of rabbitmuscle proteasomes.

FIG. 28 shows inhibition of the PGPH activity of rabbit muscleproteasomes.

FIG. 29 shows inhibition of the chymotrypsin-like activity of humanerythrocyte proteasomes.

FIG. 30 shows the effect of II-16 treatment on chymotrypsin-mediatedcleavage of LLVY-AMC substrate.

FIG. 31 shows NF-κB/luciferase activity and cytotoxicity profiles ofII-16.

FIG. 32 shows reduction of IκBα degradation and retention ofphosphorylated IκBα by II-16 in HEK293 cells (A) and the HEK293NF-κB/Luciferase reporter clone (B).

FIG. 33 shows accumulation of cell cycle regulatory proteins, p21 andp27, by II-16 treatment of HEK293 cells (A) and the HEK293NF-κB/Luciferase reporter clone (B).

FIG. 34 shows activation of Caspase-3 by II-16 in Jurkat cells.

FIG. 35 shows PARP cleavage by II-16 in Jurkat cells.

FIG. 36 shows inhibition of LeTx-induced cytotoxicity by II-16 inRAW264.7 cells.

FIG. 37 shows the effects of II-16 treatment on PARP and Pro-Caspase 3cleavage in RPMI 8226 and PC-3 cells.

FIG. 38 shows II-16 treatment of RPMI 8226 results in a dose-dependentcleavage of PARP and Pro-Caspase 3.

FIG. 39 shows in vitro proteasome inhibition by II-16, I-17, and II-18.

FIG. 40 shows proteasomal activity in PWBL prepared from II-16 treatedmice.

FIG. 41 shows epoxomicin treatment in the PWBL assay.

FIG. 42 shows intra-assay comparison.

FIG. 43 shows decreased plasma TNF levels in mice treated with LPS.

FIG. 44 depicts assay results showing the effect of Formula II-2,Formula II-3 and Formula II-4 on NF-κB mediated luciferase activity inHEK293 NF-κB/Luc Cells.

FIG. 45 depicts assay results showing the effect of Formula II-5A andFormula II-5B on NF-κB mediated luciferase activity in HEK293 NF-κB/LucCells

FIG. 46 depicts assay results showing the effect of Formula II-2,Formula II-3, and Formula II-4 on the chymotrypsin-like activity ofrabbit 20S proteasome.

FIG. 47 depicts the effect of Formula II-5A and Formula II-5B on thechymotrypsin-like activity of rabbit 20S proteasome.

FIG. 48 depicts the effect of Formulae II-2, II-3, and II-4 againstLeTx-mediated cytotoxicity.

FIG. 49 depicts the 1H NMR spectrum of a compound having structureFormula II-17.

FIG. 50 depicts the 1H NMR spectrum of a compound having structureFormula II-18.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Numerous references are cited herein. The references cited herein,including the U.S. patents cited herein, are each to be consideredincorporated by reference in their entirety into this specification.

Embodiments of the invention include, but are not limited to, providinga method for the preparation of compounds, including compounds, forexample, those described herein and analogs thereof, and to providing amethod for producing pharmaceutically acceptable anti-microbial,anti-cancer, and anti-inflammatory compositions, for example. Themethods can include the compositions in relatively high yield, whereinthe compounds and/or their derivatives are among the active ingredientsin these compositions. Other embodiments relate to providing novelcompounds not obtainable by currently available methods. Furthermore,embodiments relate to methods of treating cancer, inflammation, andinfectious diseases, particularly those affecting humans. The methodsmay include, for example, the step of administering an effective amountof a member of a class of new compounds. Preferred embodiments relate tothe compounds and methods of making and using such compounds disclosedherein, but not necessarily in all embodiments of the present invention,these objectives are met.

For the compounds described herein, each stereogenic carbon may be of Ror S configuration. Although the specific compounds exemplified in thisapplication may be depicted in a particular configuration, compoundshaving either the opposite stereochemistry at any given chiral center ormixtures thereof are also envisioned. When chiral centers are found inthe derivatives of this invention, it is to be understood that thecompounds encompasses all possible stereoisomers.

Compounds of Formula I

Some embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula I:

In certain embodiments the substituent(s) R₁, R₂, and R₃ separately mayinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variant of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl (including forexample, cyclohexylcarbinol), cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, cycloalkylacyl,hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, andhalogenated alkyl including polyhalogenated alkyl. Further, in certainembodiments, each of E₁, E₂, E₃ and E₄ may be a substituted orunsubstituted heteroatom, for example, a heteroatom separately selectedfrom the group consisting of nitrogen, sulfur and oxygen.

In some embodiments n may be equal to 1 or equal to 2. When n is equalto 2, the substituents can be the same or can be different. Furthermore,in some embodiments R₃ is not a hydrogen.

Preferably, R₂ may be a formyl. For example, the compound may have thefollowing structure I-1:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the structure of Formula I-l may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, R₂ may be a carbinol. For example, the compound may have thefollowing structure I-2:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

As an example, the structure of Formula I-2 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

As exemplary compound of Formula I may be the compound having thefollowing structure I-3:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The compound of Formula I-3 may have the following stereochemicalstructure:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Another exemplary compound Formula I may be the compound having thefollowing structure I-4:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the compound of Formula I-4 may have the followingstereochemical structure:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Still a further exemplary compound of Formula I is the compound havingthe following structure I-5:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

For example, the compound of Formula I-5 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

In some embodiments, R₂ of Formula I may be, for example, a3-methylenecyclohexene. For example, the compound may have the followingstructure of Formula I-6:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Preferably, the compound of Formula I-6 may have the followingstereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

In other embodiments, R₂ may be a cyclohexylalkylamine.

Also, in other embodiments, R₂ may be a C-Cyclohexyl-methyleneamine. Inothers, R₂ may be a cyclohexanecarbaldehyde O-oxime.

Furthermore, in some embodiments, R₂ may be a cycloalkylacyl.

Compounds of Formula II

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula II:

In certain embodiments the substituent(s) R₁, R₃, and R₄ separately mayinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variant of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, cycloalkylacyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl.Further, in certain embodiments, each of E₁, E₂, E₃ and E₄ may be asubstituted or unsubstituted heteroatom, for example, a heteroatom orsubstituted heteroatom selected from the group consisting of nitrogen,sulfur and oxygen.

In some embodiments n may be equal to 1, while in others it may be equalto 2. When n is equal to 2, the substituents can be the same or can bedifferent. Furthermore, in some embodiments R₃ is not a hydrogen. m canbe equal to 1 or 2, and when m is equal to 2, R₄ can be the same ordifferent.

E₅ may be, for example, OH, O, OR₁₀, S, SR₁₁, SO₂R₁₁, NH, NH₂, NOH,NHOH, NR₁₂, and NHOR₁₃, wherein R₁₀₋₁₃ may separately include, forexample, hydrogen, a substituted or unsubstituted of any of thefollowing: alkyl, an aryl, a heteroaryl, and the like. Also, R₁ may beCH₂CH₂X, wherein X may be, for example, H, F, Cl, Br, and I. R₃ may bemethyl. Furthermore, R₄ may include a cyclohexyl. Also, each of E₁, E₃and E₄ may be O and E₂ may be NH. Preferably, R₁ may be CH₂CH₂X, whereinX is selected from the group consisting of H, F, Cl Br, and I; whereinR₄ may include a cyclohexyl; wherein R₃ may be methyl; and wherein eachof E₁, E₃ and E₄ separately may be O and E₂ may be NH.

For example, an exemplary compound of Formula II has the followingstructure II-1:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry may be as follows:

In preferred embodiments, the compound of Formula II has any of thefollowing structures:

The following is exemplary stereochemistry for compounds having thestructures II-2, II-3, and II-4, respectively:

In other embodiments wherein R₄ may include a7-oxa-bicyclo[4.1.0]hept-2-yl). An exemplary compound of Formula II isthe following structure II-5:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following are examples of compounds having the structure of FormulaII-5:

In still further embodiments, at least one R₄ may include a subsitutedor an unsubstituted branched alkyl. For example, a compound of FormulaII may be the following structure II-6:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-6:

As another example, the compound of Formula II may be the followingstructure II-7:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-7:

In other embodiments, at least one R₄ may be a cycloalkyl and E₅ may bean oxygen. An exemplary compound of Formula II may be the followingstructure II-8:

R₈ may include, for example, hydrogen (II-8A), fluorine (II-8B),chlorine (II-8C), bromine (II-8D) and iodine (II-8E).

The following is exemplary stereochemistry for a compound having thestructure of Formula II-8:

In some embodiments E5 may be an amine oxide, giving rise to an oxime.An exemplary compound of Formula II has the following structure II-9:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine; R may be hydrogen, and a substituted or unsubstituted alkyl,aryl, or heteroaryl, and the like.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-9:

A further exemplary compound of Formula II has the following structureII-10:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-10:

In some embodiments, E₅ may be NH₂. An exemplary compound of Formula IIhas the following structure II-11:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-11:

In some embodiments, at least one R₄ may include a cycloalkyl and E₅ maybe NH₂. An exemplary compound of Formula II has the following structureII-12:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-12:

A further exemplary compound of Formula II has the following structureII-13:

R₈ may include, for example, hydrogen (II-13A), fluorine (II-13B),chlorine (II-13C), bromine (II-13D) and iodine (II-13E).

The following is exemplary stereochemistry for a compound having thestructure of Formula II-13 :

A still further exemplary compound of Formula II has the followingstructure II-14:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

The following is exemplary stereochemistry for a compound having thestructure of Formula II-14:

In some embodiments, the compounds of Formula II, may include as R₄ atleast one cycloalkene, for example. Furthermore, in some embodiments,the compounds may include a hydroxy at E₅, for example. A furtherexemplary compound of Formula II has the following structure II-15:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry may be as follows:

The following is exemplary stereochemistry for compounds having thestructures II-16, II-17, II-18, and II-19, respectively:

The compounds of Formulae II-16, II-17, II-18 and II-19 may be obtainedby fermentation, synthesis, or semi-synthesis and isolated/purified asset forth below. Furthermore, the compounds of Formulae II-16, II-17,II-18 and II-19 may be used, and are referred to, as “startingmaterials” to make other compounds described herein.

In some embodiments, the compounds of Formula II, may include a methylgroup as R₁, for example. A further exemplary compound, Formula II-20,has the following structure and stereochemistry:

In some embodiments, the compounds of Formula II, may includehydroxyethyl as R₁, for example. A further exemplary compound, FormulaII-21, has the following structure and stereochemistry:

In some embodiments, the hydroxyl group of Formula II-21 may beesterified such that R₁ may include ethylpropionate, for example. Anexemplary compound, Formula II-22, has the following structure andstereochemistry:

In some embodiments, the compounds of Formula II may include an ethylgroup as R₃, for example. A further exemplary compound of Formula II hasthe following structure II-23:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine. Exemplary stereochemistry may be as follows:

In some embodiments, the compounds of Formula II-23 may have thefollowing structure and stereochemistry, exemplified by Formula II-24C,where R₈ is chlorine:

In some embodiments, the compounds of Formula II-15 may have thefollowing stereochemistry, exemplified by the compound of Formula II-25,where R₈ is chlorine:

Compounds of Formula III

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula III:

In certain embodiments, the substituent(s) R₁ separately may include,for example, a hydrogen, a halogen, a mono-substituted, apoly-substituted or an unsubstituted variant of the following residues:saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl,acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy,nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl,carboxy, cyano, and halogenated alkyl including polyhalogenated alkyl.For example, n can be equal to 1 or 2.

In certain embodiments, R₄ may be, for example, a hydrogen, a halogen, amono-substituted, a poly-substituted or an unsubstituted variants of thefollowing residues: saturated C₁-C₂₄ alkyl, unsaturated C₂-C₂₄ alkenylor C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkylincluding polyhalogenated alkyl. In some embodiments m can be equal to 1or 2, and where m is equal to 2, the substituents can the same ordifferent. Also, each of E₁, E₂, E₃, E₄ and E₅ may be, for example, asubstituted or unsubstituted heteroatom. For example, the heteroatom maybe nitrogen, sulfur or oxygen.

Compounds of Formula IV

Other embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula IV:

In certain embodiments, the substituent(s) R₁ R₃, and R₅ may separatelyinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted oran unsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, andhalogenated alkyl including polyhalogenated alkyl. Also, each of E₁, E₂,E₃, E₄ and E₅ may be a heteroatom or substituted heteroatom, forexample, nitrogen, sulfur or oxygen. In some embodiments, R₃ is not ahydrogen. n is equal to 1 or 2. When n is equal to 2, the substituentscan be the same or can be different. Also, m can be 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 1 0, or 11. When m is greater than 1, the substituents can bethe same or different.

In some embodiments R₅ may give rise to a di-substituted cyclohexyl. Anexemplary compound of Formula IV is the following structure IV-1, withand without exemplary stereochemistry:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine. The substituent(s) R₆ and R₇ may separately include a hydrogen,a halogen, a mono-substituted, a poly-substituted or an unsubstitutedvariants of the following residues: saturated C₁-C₄ alkyl, unsaturatedC₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy,aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino,aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy,alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and halogenated alkylincluding polyhalogenated alkyl. Further, R₆ and R₇ both may be the sameor different.

For example, an exemplary compound of Formula IV has the followingstructure IV-2:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry may be as follows:

For example, an exemplary compound of Formula IV has the followingstructure IV-3:

R₈ may include, for example, hydrogen (IV-3A), fluorine (IV-3B),chlorine (IV-3C), bromine (IV-3D) and iodine (WV-3E).

Exemplary structure and stereochemistry may be as follows:

Additional exemplary structure and stereochemistry may be as follows:

For example, an exemplary compound of Formula IV has the followingstructure IV-4:

R₈ may include, for example, hydrogen, fluorine, chlorine, bromine andiodine.

Exemplary stereochemistry may be as follows:

Compounds of Formula V

Some embodiments provide compounds, and methods of producing a class ofcompounds, pharmaceutically acceptable salts and pro-drug estersthereof, wherein the compounds are represented by Formula V:

In certain embodiments, the substituent(s) R₁ and R₅ may separatelyinclude a hydrogen, a halogen, a mono-substituted, a poly-substituted orunsubstituted variants of the following residues: saturated C₁-C₂₄alkyl, unsaturated C₂-C₂₄ alkenyl or C₂-C₂₄ alkynyl, acyl, acyloxy,alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl,alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido,phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, andhalogenated alkyl including polyhalogenated alkyl. In certainembodiments, each of E₁, E₂, E₃, E₄ and E₅ may be a heteroatom orsubstituted heteroatom, for example, nitrogen, sulfur or oxygen. n canbe equal to 1 or 2, and when n is equal to 2, the substituents can bethe same or different. Preferably, m may be, for example, 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or 11. When m is greater than 1, R₅ may be the sameor different.

Certain embodiments also provide pharmaceutically acceptable salts andpro-drug esters of the compound of Formulae I-V, and provide methods ofobtaining and purifying such compounds by the methods disclosed herein.

The term “pro-drug ester,” especially when referring to a pro-drug esterof the compound of Formula I synthesized by the methods disclosedherein, refers to a chemical derivative of the compound that is rapidlytransformed in vivo to yield the compound, for example, by hydrolysis inblood or inside tissues. The term “pro-drug ester” refers to derivativesof the compounds disclosed herein formed by the addition of any ofseveral ester-forming groups that are hydrolyzed under physiologicalconditions. Examples of pro-drug ester groups include pivoyloxymethyl,acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as othersuch groups known in the art, including a(5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drugester groups can be found in, for example, T. Higuchi and V. Stella, in“Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series,American Chemical Society (1975); and “Bioreversible Carriers in DrugDesign: Theory and Application”, edited by E. B. Roche, Pergamon Press:New York, 14-21 (1987) (providing examples of esters useful as prodrugsfor compounds containing carboxyl groups). Each of the above-mentionedreferences is hereby incorporated by reference in its entirety.

The term “pro-drug ester,” as used herein, also refers to a chemicalderivative of the compound that is rapidly transformed in vivo to yieldthe compound, for example, by hydrolysis in blood.

The term “pharmaceutically acceptable salt,” as used herein, andparticularly when referring to a pharmaceutically acceptable salt of acompound, including Formulae I-V, and Formula I-V as produced andsynthesized by the methods disclosed herein, refers to anypharmaceutically acceptable salts of a compound, and preferably refersto an acid addition salt of a compound. Preferred examples ofpharmaceutically acceptable salt are the alkali metal salts (sodium orpotassium), the alkaline earth metal salts (calcium or magnesium), orammonium salts derived from ammonia or from pharmaceutically acceptableorganic amines, for example C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.With respect to compounds synthesized by the method of this embodimentthat are basic amines, the preferred examples of pharmaceuticallyacceptable salts are acid addition salts of pharmaceutically acceptableinorganic or organic acids, for example, hydrohalic, sulfuric,phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid,for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic,nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonicacid.

Preferred pharmaceutical compositions disclosed herein includepharmaceutically acceptable salts and pro-drug esters of the compound ofFormulae I-V obtained and purified by the methods disclosed herein.Accordingly, if the manufacture of pharmaceutical formulations involvesintimate mixing of the pharmaceutical excipients and the activeingredient in its salt form, then it is preferred to use pharmaceuticalexcipients which are non-basic, that is, either acidic or neutralexcipients.

It will be also appreciated that the phrase “compounds and compositionscomprising the compound,” or any like phrase, is meant to encompasscompounds in any suitable form for pharmaceutical delivery, as discussedin further detail herein. For example, in certain embodiments, thecompounds or compositions comprising the same may include apharmaceutically acceptable salt of the compound.

In one embodiment the compounds may be used to treat microbial diseases,cancer, and inflammation. Disease is meant to be construed broadly tocover infectious diseases, and also autoimmune diseases, non-infectiousdiseases and chronic conditions. In a preferred embodiment, the diseaseis caused by a microbe, such as a bacterium, a fungi, and protozoa, forexample. The methods of use may also include the steps of administeringa compound or composition comprising the compound to an individual withan infectious disease or cancer. The compound or composition can beadministered in an amount effective to treat the particular infectiousdisease, cancer or inflammatory condition.

The infectious disease may be, for example, one caused by Bacillus, suchas B. anthracis and B. cereus. The infectious disease may be one causedby a protozoa, for example, a Leishmania, a Plasmodium or a Trypanosoma.The compound or composition may be administered with a pharmaceuticallyacceptable carrier, diluent, excipient, and the like.

The cancer may be, for example, a multiple myeloma, a colorectalcarcinoma, a prostate carcinoma, a breast adenocarcinoma, a non-smallcell lung carcinoma, an ovarian carcinoma, a melanoma, and the like.

The inflammatory condition may be, for example, rheumatoid arthritis,asthma, multiple sclerosis, psoriasis, stroke, myocardial infarction,and the like.

The term “halogen atom,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,i.e., fluorine, chlorine, bromine, or iodine, with bromine and chlorinebeing preferred.

The term “alkyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, saturated hydrocarbon, with C₁-C₆unbranched, saturated, unsubstituted hydrocarbons being preferred, withmethyl, ethyl, isobutyl, and tert-butylpropyl, and pentyl being mostpreferred. Among the substituted, saturated hydrocarbons, C₁-C₆ mono-and di- and per-halogen substituted saturated hydrocarbons andamino-substituted hydrocarbons are preferred, with perfluromethyl,perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl beingthe most preferred.

The term “substituted” has its ordinary meaning, as found in numerouscontemporary patents from the related art. See, for example, U.S. Pat.Nos. 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443;and 6,350,759; all of which are incorporated herein in their entiretiesby reference. Specifically, the definition of substituted is as broad asthat provided in U.S. Pat. No. 6,509,331, which defines the term“substituted alkyl” such that it refers to an alkyl group, preferably offrom 1 to 10 carbon atoms, having from 1 to 5 substituents, andpreferably 1 to 3 substituents, selected from the group consisting ofalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano,halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Theother above-listed patents also provide standard definitions for theterm “substituted” that are well-understood by those of skill in theart.

The term “cycloalkyl” refers to any non-aromatic hydrocarbon ring,preferably having five to twelve atoms comprising the ring. The term“acyl” refers to alkyl or aryl groups derived from an oxoacid, with anacetyl group being preferred.

The term “alkenyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, unsaturated hydrocarbon includingpolyunsaturated hydrocarbons, with C₁-C₆ unbranched, mono-unsaturatedand di-unsaturated, unsubstituted hydrocarbons being preferred, andmono-unsaturated, di-halogen substituted hydrocarbons being mostpreferred. The term “cycloalkenyl” refers to any non-aromatichydrocarbon ring, preferably having five to twelve atoms comprising thering.

The terms “aryl,” “substituted aryl,” “heteroaryl,” and “substitutedheteroaryl,” as used herein, refer to aromatic hydrocarbon rings,preferably having five, six, or seven atoms, and most preferably havingsix atoms comprising the ring. “Heteroaryl” and “substitutedheteroaryl,” refer to aromatic hydrocarbon rings in which at least oneheteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring alongwith at least one carbon atom. The term “heterocycle” or “heterocyclic”refer to any cyclic compound containing one or more heteroatoms. Thesubstituted aryls, heterocycles and heteroaryls can be substituted withany substituent, including those described above and those known in theart.

The term “alkoxy” refers to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ether, with C₁-C₆ unbranched,saturated, unsubstituted ethers being preferred, with methoxy beingpreferred, and also with dimethyl, diethyl, methyl-isobutyl, andmethyl-tert-butyl ethers also being preferred. The term “cycloalkoxy”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring. The term “alkoxy carbonyl” refers toany linear, branched, cyclic, saturated, unsaturated, aliphatic oraromatic alkoxy attached to a carbonyl group. The examples includemethoxycarbonyl group, ethoxycarbonyl group, propyloxycarbonyl group,isopropyloxycarbonyl group, butoxycarbonyl group, sec-butoxycarbonylgroup, tert-butoxycarbonyl group, cyclopentyloxycarbonyl group,cyclohexyloxycarbonyl group, benzyloxycarbonyl group, allyloxycarbonylgroup, phenyloxycarbonyl group, pyridyloxycarbonyl group, and the like.

The terms “pure,” “purified,” “substantially purified,” and “isolated”as used herein refer to the compound of the embodiment being free ofother, dissimilar compounds with which the compound, if found in itsnatural state, would be associated in its natural state. In certainembodiments described as “pure,” “purified,” “substantially purified,”or “isolated” herein, the compound may comprise at least 0.5%, 1%, 5%,10%, or 20%, and most preferably at least 50% or 75% of the mass, byweight, of a given sample.

The terms “derivative,” “variant,” or other similar term refers to acompound that is an analog of the other compound.

Certain of the compounds of Formula I-V may be obtained and purified ormay be obtained via semi-synthesis from purified compounds as set forthherein. Generally, without being limited thereto, the compounds ofFormula II-15, preferably, Formulae II-16, II-17, II-18 and II-19, maybe obtained synthetically or by fermentation. Exemplary fermentationprocedures are provided below. Futher, the compounds of Formula II-15,preferably, Formulae II-16, II-17, II-18 and II-19 may be used asstarting compounds in order to obtain/synthesize various of the othercompounds described herein. Exemplary non-limiting syntheses areprovided herein.

Formula II-16 is currently produced through a high-yield salinefermentation (˜200 mg/L) and modifications of the conditions has yieldednew analogs in the fermentation extracts. FIG. 1 shows the chemicalstructure of II-16. Additional analogs can be generated through directedbiosynthesis. Directed biosynthesis is the modification of a naturalproduct by adding biosynthetic precursor analogs to the fermentation ofproducing microorganisms (Lam, et al., J Antibiot (Tokyo) 44:934 (1991),Lam, et al., J Antibiot (Tokyo) 54:1 (2001); which is herebyincorporated by reference in its entirety).

Exposing the producing culture to analogs of acetic acid, phenylalanine,valine, butyric acid, shikimic acid, and halogens, preferably, otherthan chlorine, can lead to the formation of new analogs. The new analogsproduced can be easily detected in crude extracts by HPLC and LC-MS. Forexample, after manipulating the medium with different concentrations ofsodium bromide, a bromo-analog, Formula II-18, was successfully producedin shake-flask culture at a titer of 14 mg/L.

A second approach to generate new analogs is through biotransformation.Biotransformation reactions are chemical reactions catalyzed by enzymesor whole cells containing these enzymes. Zaks, A., Curr Opin Chem Biol5:130 (2001). Microbial natural products are ideal substrates forbiotransformation reactions as they are synthesized by a series ofenzymatic reactions inside microbial cells. Riva, S., Curr Opin ChemBiol 5:106 (2001).

Given the structure of the described compounds, including those ofFormula II-15, for example, the possible biosynthetic origins areacetyl-CoA, ethylmalonyl-CoA, phenylalanine and chlorine.Ethylmalonyl-CoA is derived from butyryl-CoA, which can be derivedeither from valine or crotonyl-CoA. Liu, et al., Metab Eng 3:40 (2001).Phenylalanine is derived from shikimic acid.

Production of Compounds of Formulae II-16, II-17, and II-18

The production of compounds of Formulae II-16, II-17, and II-18 may becarried out by cultivating strain CNB476 in a suitable nutrient mediumunder conditions described herein, preferably under submerged aerobicconditions, until a substantial amount of compounds are detected in thefermentation; harvesting by extracting the active components from thefermentation broth with a suitable solvent; concentrating the solventcontaining the desired components; then subjecting the concentratedmaterial to chromatographic separation to isolate the compounds fromother metabolites also present in the cultivation medium.

FIG. 2 shows some collection sites worldwide for the culture (CNB476),which is also refered to as Salinospora. FIG. 3 shows colonies ofSalinospora. FIG. 4 shows the typical 16S rDNA sequence of theSalinospora. Bars represent characteristic signature nucleotides of theSalinospora that separate them from their nearest relatives.

The culture (CNB476) was deposited on Jun. 20, 2003 with the AmericanType Culture Collection (ATCC) in Rockville, Md. and assigned the ATCCpatent deposition number PTA-5275. The ATCC deposit meets all of therequirements of the Budapest treaty. The culture is also maintained atand available from Nereus Pharmaceutical Culture Collection at 10480Wateridge Circle, San Diego, Calif. 92121. In addition to the specificmicroorganism described herein, it should be understood that mutants,such as those produced by the use of chemical or physical mutagensincluding X-rays, etc. and organisms whose genetic makeup has beenmodified by molecular biology techniques, may also be cultivated toproduce the starting compounds of Formulae II-16, II-17, and II-18.

Fermentation of Strain CNB476

Production of compounds can be achieved at temperature conducive tosatisfactory growth of the producing organism, e.g. from 16 degree C. to40 degree C., but it is preferable to conduct the fermentation at 22degree C. to 32 degree C. The aqueous medium can be incubated for aperiod of time necessary to complete the production of compounds asmonitored by high pressure liquid chromatography (HPLC), preferably fora period of about 2 to 10 days, on a rotary shaker operating at about 50rpm to 400 rpm, preferably at 150 rpm to 250 rpm, for example.

Growth of the microorganisms may be achieved by one of ordinary skill ofthe art by the use of appropriate medium. Broadly, the sources of carboninclude glucose, fructose, mannose, maltose, galactose, mannitol andglycerol, other sugars and sugar alcohols, starches and othercarbohydrates, or carbohydrate derivatives such as dextran, cerelose, aswell as complex nutrients such as oat flour, corn meal, millet, corn,and the like. The exact quantity of the carbon source that is utilizedin the medium will depend in part, upon the other ingredients in themedium, but an amount of carbohydrate between 0.5 to 25 percent byweight of the medium can be satisfactorily used, for example. Thesecarbon sources can be used individually or several such carbon sourcesmay be combined in the same medium, for example. Certain carbon sourcesare preferred as hereinafter set forth.

The sources of nitrogen include amino acids such as glycine, arginine,threonine, methionine and the like, ammonium salt, as well as complexsources such as yeast extracts, corn steep liquors, distiller solubles,soybean meal, cotttonseed meal, fish meal, peptone, and the like. Thevarious sources of nitrogen can be used alone or in combination inamounts ranging from 0.5 to 25 percent by weight of the medium, forexample.

Among the nutrient inorganic salts, which can be incorporated in theculture media, are the customary salts capable of yielding sodium,potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included are trace metals such as cobalt, manganese,iron, molybdenum, zinc, cadmium, and the like.

Biological Activity and Uses of Compounds

Some embodiments relate to methods of treating cancer, inflammation, andinfectious diseases, particularly those affecting humans. The methodsmay include, for example, the step of administering an effective amountof a member of a class of new compounds. Thus, the compounds disclosedherein may be used to treat cancer, inflammation, and infectiousdisease.

The compounds have various biological activities. For example, thecompounds have chemosensitizing activity, anti-microbial,anti-inflammation, and anti-cancer activity.

The compounds have proteasome inhibitory activity. The proteasomeinhibitory activity may, in whole or in part, contribute to the abilityof the compounds to act as anti-cancer, anti-inflammatory, andanti-microbial agents.

The proteasome is a multisubunit protease that degrades intracellularproteins through its chymotrypsin-like, trypsin-like andpeptidylglutamyl-peptide hydrolyzing (PGPH; and also know as thecaspase-like activity) activities. The 26S proteasome contains aproteolytic core called the 20S proteasome and two 19S regulatorysubunits. The 20S proteasome is responsible for the proteolytic activityagainst many substrates including damaged proteins, the transcriptionfactor NF-κB and its inhibitor IκB, signaling molecules, tumorsuppressors and cell cycle regulators. There are three distinct proteaseactivities within the proteasome: 1) chymotrypsin-like; 2) trypsin-like;and the 3) peptidyl glutamyl peptide hydrolyzing (PGPH) activity.

As an example, compounds of Formula II-16 were more potent (EC₅₀ 2 nM)at inhibiting the chymotrypsin-like activity of rabbit muscleproteasomes than Omuralide (EC₅₀ 52 nM) and also inhibited thechymotrypsin-like activity of human erythrocyte derived proteasomes(EC₅₀ ˜250 pM). FIG. 5 shows omuralide, which is a degradation productof Lactacystin, and it shows a compound of Formula II-16. Compounds ofFormula II-16 exhibit a significant preference for inhibitingchymotrypsin-like activity of the proteasome over inhibiting thecatalytic activity of chymotrypsin. Compounds of Formula II-16 alsoexhibit low nM trypsin-like inhibitory activity (˜10 nM), but are lesspotent at inhibiting the PGPH activity of the proteasome (EC₅₀ ˜350 nM).

Additional studies have characterized the effects of compounds describedherein, including studies of Formula II-16 on the NF-κB/IκB signalingpathway. Treatment of HEK293 cells (human embryonic kidney) with TumorNecrosis Factor-alpha (TNF-α) induces phosphorylation andproteasome-mediated degradation of IκBα: followed by NF-κB activation.To confirm proteasome inhibition, HEK293 cells were pre-treated for 1hour with compounds of Formula II-16 followed by TNF-α stimulation.Treatment with compounds of Formula II-16 promoted the accumulation ofphosphorylated IκBα suggesting that the proteasome-mediated IκBαdegradation was inhibited.

Furthermore, a stable HEK293 clone (NF-κB/Luc 293) was generatedcarrying a luciferase reporter gene under the regulation of 5× NF-κBbinding sites. Stimulation of NF-κB/Luc 293 cells with TNF-α increasesluciferase activity as a result of NF-κB activation while pretreatmentwith compounds of Formula II-16 decreases activity. Western blotanalyses demonstrated that compounds of Formula II-16 promoted theaccumulation of phosphorylated-IκBα and decreased the degradation oftotal IκBα in the NF-κB/Luc 293 cells. Compounds of Formula II-16 werealso shown to increase the levels of the cell cycle regulatory proteins,p21 and p27.

Tumor cells may be more sensitive to proteasome inhibitors than normalcells. Moreover, proteasome inhibition increases the sensitivity ofcancer cells to anticancer agents. The cytotoxic activity of thecompounds described herein, including Formula II-16, were examined forcytotoxic activity against various cancer cell lines. Formula II-16 wasexamined, for example, in the National Cancer Institute screen of 60human tumor cell lines. Formula II-16 exhibited selective cytotoxicactivity with a mean GI₅₀ value (the concentration to achieve 50% growthinhibition) of less than 10 nM. The greatest potency was observedagainst SK-MEL-28 melanoma and MDA-MB-235 breast cancer cells [both withLC₅₀ (the concentration with 50% cell lethality) <10 nM].

A panel of cell lines including human colorectal (HT-29 and LoVo),prostate (PC3), breast (MDA-MB-23 1), lung (NCI-H292), ovarian (OVCAR3),acute T-cell leukemia (Jurkat), murine melanoma (B16-F10) and normalhuman fibroblasts (CCD-27sk) was treated with Salinosporamide A for 48hto assess cytotoxic activity. HT-29, LoVo, PC3, MDA-MB-231, NCI-H292,OVCAR3, Jurkat, and B16-F10 cells were sensitive with EC₅₀ values of 47,69, 78, 67, 97, 69, 10, and 33 nM, respectively. In contrast, the EC₅₀values for CCD-27sk cells were 196 nM. Treatment of Jurkat cells withSalinosporamide A at the approximate EC₅₀ resulted in Caspase-3activation and cleavage of PARP confirming the induction of apoptosis.

The anti-anthrax activity of the described compounds was evaluated usingan in vitro LeTx induced cytotoxicity assay. As one example, the resultsindicate that Formula II-16 is a potent inhibitor of LeTx-inducedcytotoxicity of murine macrophage-like RAW264.7 cells. Treatment ofRAW264.7 cells with Formula II-16 resulted in a 10-fold increase in theviability of LeTx-treated cells compared to LeTx treatment alone(average EC₅₀ of <4 nM).

Potential Chemosensitizing Effects of Formula II-16

Additional studies have characterized the effects of the compoundsdescribed herein on the NF-κB/IκB signaling pathway (see the Examples).In unstimulated cells, the transcription factor nuclear factor-kappa B(NF-?B) resides in the cytoplasm in an inactive complex with theinhibitory protein IκB (inhibitor of NF-κB). Various stimuli can causeI?B phosphorylation by I?B kinase, followed by ubiquitination anddegradation by the proteasome. Following the degradation of I?B, NF-?Btranslocates to the nucleus and regulates gene expression, affectingmany cellular processes including inhibition of apoptosis. Chemotherapyagents such as CPT-11 (Irinotecan) can activate NF-?B in human coloncancer cell lines including LoVo cells, resulting in a decreased abilityof these cells to undergo apoptosis. Painter, R. B. Cancer Res 38:4445(1978). Velcadey™ is a dipeptidyl boronic acid that inhibits thechymotrypsin-like activity of the proteasome (Lightcap, et al., ClinChem 46:673 (2000), Adams, et al., Cancer Res 59:2615 (1999), Adams,Curr Opin Oncol 14:628 (2002)) while enhancing the trypsin and PGPHactivities. Recently approved as a proteasome inhibitor, Velcade™,(PS-341; Millennium Pharmaceuticals, Inc.) has been shown to be directlytoxic to cancer cells and also enhance the cytotoxic activity of CPT-11in LoVo cells in vitro and in a LoVo xenograft model by inhibiting I?Bdegradation by the proteasome. Blum, et al., Ann Intern Med 80:249(1974). In addition, Velcade™ was found to inhibit the expression ofproangiogenic chemokines/cytokines Growth Related Oncogene-alpha (GRO-α)and Vascular Endothelial Growth Factor (VEGF) in squamous cellcarcinoma, presumably through inhibition of the NF-κB pathway. Dick, etal., J Biol Chem 271:7273 (1996). These data suggest that proteasomeinhibition may not only decrease tumor cell survival and growth, butalso angiogenesis.

Anti-Anthrax Activity

Another potential application for proteasome inhibitors comes fromrecent studies on the biodefense Category A agent B. anthracis(anthrax). Anthrax spores are inhaled and lodge in the lungs where theyare ingested by macrophages. Within the macrophage, spores germinate,the organism replicates, resulting ultimately in killing of the cell.Before killing occurs, however, infected macrophages migrate to thelymph nodes where, upon death, they release their contents allowing theorganism to enter the bloodstream, further replicate, and secrete lethaltoxins. Hanna, et al., Proc Natl Acad Sci U S A 90:10198 (1993). Anthraxtoxins are responsible for the symptoms associated with anthrax. Twoproteins that play a key role in the pathogenesis of anthrax areprotective antigen (PA, 83 kDa) and lethal factor (LF, 90 kDa) which arecollectively known as lethal toxin (LeTx). LF has an enzymatic function,but requires PA to achieve its biological effect. Neither PA or LF causedeath individually; however, when combined they cause death wheninjected intravenously in animals. Kalns, et al., Biochem Biophys ResCommun 297:506 (2002), Kalns, et al., Biochem Biophys Res Commun 292:41(2002).

Protective antigen, the receptor-binding component of anthrax toxin, isresponsible for transporting lethal factor into the host cell. PAoligomerizes into a ring-shaped heptamer (see FIG. 6). Each heptamer,bound to its receptor on the surface of a cell, has the ability to bindup to three molecules of LF. The complex formed between the PA heptamerand LF is taken into the cell by receptor-mediated endocytosis.Following endocytosis, LF is released into the cytosol where it attacksvarious cellular targets. Mogridge, et al., Biochemistry 41:1079 (2002),Lacy, et al., J Biol Chem 277:3006 (2002), Bradley, et al., Nature414:225 (2001).

Lethal factor is a zinc dependent metalloprotease, which in the cytosolcan cleave and inactivate signaling proteins of the mitogen-activatedprotein kinase kinase family (MAPKK). Duesbery, et al., Science 280:734(1998), Bodart, et al., Cell Cycle 1:10 (2002), Vitale, et al., J ApplMicrobiol 87:288 (1999), Vitale, et al., Biochem J 352 Pt 3:739 (2000).Of the seven different known MAPK kinases, six have been shown to becleaved by LF. Within the cell, MAPK kinase pathways transduce varioussignals involved in cell death, proliferation, and differentiationmaking these proteins highly significant targets. However, certaininhibitors that prevent LeTx-induced cell death, do not prevent MAPKKcleavage by LF suggesting that this activity is not sufficient forinduction of cell death. Kim, et al., J Biol Chem 278:7413 (2003), Lin,et al., Curr Microbiol 33:224 (1996).

Studies have suggested that inhibition of the proteasome can preventLeTx-induced cell death. Tang, et al., Infect Immun 67:3055 (1999). Datahave shown that proteasome activity is required for LeTx-mediatedkilling of RAW264.7 macrophage-like cells and that proteasome inhibitorsprotect RAW264.7 cells from LeTx. Proteasome inhibition did not blockMEK1 cleavage, suggesting the LeTx pathway is not blocked upstream ofMEK1 cleavage in these studies. Additionally, there is no increase inproteasome activity in cells treated with LeTx. These data suggestedthat a novel, potent proteasome inhibitor like the compounds describedherein, may also prevent LeTx-induced cell death as illustrated in FIG.6.

The receptor for PA has been identified and is expressed by many celltypes. Escuyer, et al., Infect Immun 59:3381 (1991). Lethal toxin isactive in a few cell culture lines of macrophages causing cell deathwithin a few hours. Hanna, et al., Proc Natl Acad Sci USA 90:10198(1993), Kim, et al., J Biol Chem 278:7413 (2003), Lin, et al., CurrMicrobiol 33:224 (1996). LeTx can induce both necrosis and apoptosis inmouse macrophage-like RAW264.7 and J774A.1 cells upon in vitrotreatment.

The results indicate that the compounds described herein act as a potentinhibitor of LeTx-induced cytotoxicity of murine macrophage-likeRAW264.7 cells. Treatment of RAW264.7 cells with, for example, compoundsof Formula II-16, resulted in a 10-fold increase in the viability ofLeTx-treated cells compared to LeTx treatment alone (average EC₅₀ of <4nM) and therefore provide a valuable therapy for anthrax infections.Formula II-16, for example, promoted survival of RAW264.7macrophage-like cells in the presence of LeTx indicating that thiscompound and its derivatives provide a valuable clinical therapeutic foranthrax infection.

Pharmaceutical Compositions

In one embodiment, the compounds disclosed herein are used inpharmaceutical compositions. The compounds preferably can be produced bythe methods disclosed herein. The compounds can be used, for example, inpharmaceutical compositions comprising a pharmaceutically acceptablecarrier prepared for storage and subsequent administration. Also,embodiments relate to a pharmaceutically effective amount of theproducts and compounds disclosed above in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), which is incorporated herein by reference in itsentirety. Preservatives, stabilizers, dyes and even flavoring agents maybe provided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. In addition, antioxidants and suspending agents may beused.

The compositions, particularly those of Formulae I-V, may be formulatedand used as tablets, capsules, or elixirs for oral administration;suppositories for rectal administration; sterile solutions, suspensionsfor injectable administration; patches for transdermal administration,and sub-dermal deposits and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), may be utilized.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations); allof which are incorporated herein by reference in their entireties.

Further disclosed herein are various pharmaceutical compositions wellknown in the pharmaceutical art for uses that include intraocular,intranasal, and intraauricular delivery. Pharmaceutical formulationsinclude aqueous ophthalmic solutions of the active compounds inwater-soluble form, such as eyedrops, or in gellan gum (Shedden et al.,Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayer et al.,Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments; ophthalmicsuspensions, such as microparticulates, drug-containing small polymericparticles that are suspended in a liquid carrier medium (Joshi, A. 1994J Ocul Pharmacol 10:29-45), lipid-soluble formulations (Alm et al.,Prog. Clin. Biol. Res., 312:447-58 (1989)), and microspheres (Mordenti,Toxicol. Sci., 52(l):101-6 (1999)); and ocular inserts. All of theabove-mentioned references, are incorporated herein by reference intheir entireties. Such suitable pharmaceutical formulations are mostoften and preferably formulated to be sterile, isotonic and buffered forstability and comfort. Pharmaceutical compositions may also includedrops and sprays often prepared to simulate in many respects nasalsecretions to ensure maintenance of normal ciliary action. As disclosedin Remington's Pharmaceutical Sciences (Mack Publishing, 18^(th)Edition), which is incorporated herein by reference in its entirety, andwell-known to those skilled in the art, suitable formulations are mostoften and preferably isotonic, slightly buffered to maintain a pH of 5.5to 6.5, and most often and preferably include anti-microbialpreservatives and appropriate drug stabilizers. Pharmaceuticalformulations for intraauricular delivery include suspensions andointments for topical application in the ear. Common solvents for suchaural formulations include glycerin and water.

When used as an anti-cancer, anti-inflammatory or anti-microbialcompound, for example, the compounds of Formulae I-V or compositionsincluding Formulae I-V can be administered by either oral or non-oralpathways. When administered orally, it can be administered in capsule,tablet, granule, spray, syrup, or other such form. When administerednon-orally, it can be administered as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like, when administered via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, or the like.

In one embodiment, the anti-cancer, anti-inflammatory or anti-microbialcan be mixed with additional substances to enhance their effectiveness.In one embodiment, the anti-microbial is combined with an additionalanti-microbial. In another embodiment, the anti-microbial is combinedwith a drug or medicament that is helpful to a patient that is takinganti-microbials.

Methods of Administration

In an alternative embodiment, the disclosed chemical compounds and thedisclosed pharmaceutical compositions are administered by a particularmethod as an anti-microbial. Such methods include, among others, (a)administration though oral pathways, which administration includesadministration in capsule, tablet, granule, spray, syrup, or other suchforms; (b) administration through non-oral pathways, whichadministration includes administration as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like; administration via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or thelike; as well as (c) administration topically, (d) administrationrectally, or (e) administration vaginally, as deemed appropriate bythose of skill in the art for bringing the compound of the presentembodiment into contact with living tissue; and (f) administration viacontrolled released formulations, depot formulations, and infusion pumpdelivery. As further examples of such modes of administration and asfurther disclosure of modes of administration, disclosed herein arevarious methods for administration of the disclosed chemical compoundsand pharmaceutical compositions including modes of administrationthrough intraocular, intranasal, and intraauricular pathways.

The pharmaceutically effective amount of the compositions that includethe described compounds, including those of Formulae I-V, required as adose will depend on the route of administration, the type of animal,including human, being treated, and the physical characteristics of thespecific animal under consideration. The dose can be tailored to achievea desired effect, but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize.

In practicing the methods of the embodiment, the products orcompositions can be used alone or in combination with one another, or incombination with other therapeutic or diagnostic agents. These productscan be utilized in vivo, ordinarily in a mammal, preferably in a human,or in vitro. In employing them in vivo, the products or compositions canbe administered to the mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, vaginally, nasally or intraperitoneally,employing a variety of dosage forms. Such methods may also be applied totesting chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired affects and thetherapeutic indication. Typically, dosages may be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively dosages may bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art. Administration is preferably oral on adaily or twice daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975, which is incorporated herein by reference in its entirety. Itshould be noted that the attending physician would know how to and whento terminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. A variety oftechniques for formulation and administration may be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990), which is incorporated herein by reference in itsentirety. Suitable administration routes may include oral, rectal,transdermal, vaginal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

For injection, the agents of the embodiment may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theembodiment into dosages suitable for systemic administration is withinthe scope of the embodiment. With proper choice of carrier and suitablemanufacturing practice, the compositions disclosed herein, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the embodiment to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration may be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions may be manufactured in amanner that is itself known, for example, by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, may be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, dogs or monkeys, may be determined using known methods. Theefficacy of a particular compound may be established using several artrecognized methods, such as in vitro methods, animal models, or humanclinical trials. Art-recognized in vitro models exist for nearly everyclass of condition, including the conditions abated by the compoundsdisclosed herein, including cancer, cardiovascular disease, and variousimmune dysfunction, and infectious diseases. Similarly, acceptableanimal models may be used to establish efficacy of chemicals to treatsuch conditions. When selecting a model to determine efficacy, theskilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, and regime. Ofcourse, human clinical trials can also be used to determine the efficacyof a compound in humans.

When used as an anti-microbial, anti-cancer, or anti-inflammatory agent,the compounds disclosed herein may be administered by either oral or anon-oral pathways. When administered orally, it can be administered incapsule, tablet, granule, spray, syrup, or other such form. Whenadministered non-orally, it can be administered as an aqueoussuspension, an oily preparation or the like or as a drip, suppository,salve, ointment or the like, when administered via injection,subcutaneously, intraperitoneally, intravenously, intramuscularly,intradermally, or the like. Controlled release formulations, depotformulations, and infusion pump delivery are similarly contemplated.

The compositions disclosed herein in pharmaceutical compositions mayalso comprise a pharmaceutically acceptable carrier. Such compositionsmay be prepared for storage and for subsequent administration.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, such compositions may be formulated and used as tablets,capsules or solutions for oral administration; suppositories for rectalor vaginal administration; sterile solutions or suspensions forinjectable administration. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients include, but are not limited to, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

The products or compositions of the embodiment, as described above, maybe used alone or in combination with one another, or in combination withother therapeutic or diagnostic agents. These products can be utilizedin vivo or in vitro. The useful dosages and the most useful modes ofadministration will vary depending upon the age, weight and animaltreated, the particular compounds employed, and the specific use forwhich these composition or compositions are employed. The magnitude of adose in the management or treatment for a particular disorder will varywith the severity of the condition to be treated and to the route ofadministration, and depending on the disease conditions and theirseverity, the compositions may be formulated and administered eithersystemically or locally. A variety of techniques for formulation andadministration may be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990).

To formulate the compounds of Formulae I-V as an anti-microbial, ananti-cancer, or an anti-inflammatory agent, known surface active agents,excipients, smoothing agents, suspension agents and pharmaceuticallyacceptable film-forming substances and coating assistants, and the likemay be used. Preferably alcohols, esters, sulfated aliphatic alcohols,and the like may be used as surface active agents; sucrose, glucose,lactose, starch, crystallized cellulose, mannitol, light anhydroussilicate, magnesium aluminate, magnesium methasilicate aluminate,synthetic aluminum silicate, calcium carbonate, sodium acid carbonate,calcium hydrogen phosphate, calcium carboxymethyl cellulose, and thelike may be used as excipients; magnesium stearate, talc, hardened oiland the like may be used as smoothing agents; coconut oil, olive oil,sesame oil, peanut oil, soya may be used as suspension agents orlubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methylacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents. In addition to the foregoing preferred ingredients,sweeteners, fragrances, colorants, preservatives and the like may beadded to the administered formulation of the compound produced by themethod of the embodiment, particularly when the compound is to beadministered orally.

The compounds and compositions may be orally or non-orally administeredto a human patient in the amount of about 0.001 mg/kg/day to about10,000 mg/kg/day of the active ingredient, and more preferably about 0.1mg/kg/day to about 100 mg/kg/day of the active ingredient at,preferably, one time per day or, less preferably, over two to about tentimes per day. Alternatively and also preferably, the compound producedby the method of the embodiment may preferably be administered in thestated amounts continuously by, for example, an intravenous drip. Thus,for the example of a patient weighing 70 kilograms, the preferred dailydose of the active or anti-infective ingredient would be about 0.07mg/day to about 700 gm/day, and more preferable, 7 mg/day to about 7grams/day. Nonetheless, as will be understood by those of skill in theart, in certain situations it may be necessary to administer theanti-cancer, anti-inflammatory or the anti-infective compound of theembodiment in amounts that excess, or even far exceed, the above-stated,preferred dosage range to effectively and aggressively treatparticularly advanced cancerss or infections.

In the case of using the anti-microbial produced by methods of theembodiment as a biochemical test reagent, the compound produced bymethods of the embodiment inhibits the progression of the disease whenit is dissolved in an organic solvent or hydrous organic solvent and itis directly applied to any of various cultured cell systems. Usableorganic solvents include, for example, methanol, methylsulfoxide, andthe like. The formulation can, for example, be a powder, granular orother solid inhibitor, or a liquid inhibitor prepared using an organicsolvent or a hydrous organic solvent. While a preferred concentration ofthe compound produced by the method of the embodiment for use as ananti-microbial, anticancer or anti-tumor compound is generally in therange of about 1 to about 100 μg/ml, the most appropriate use amountvaries depending on the type of cultured cell system and the purpose ofuse, as will be appreciated by persons of ordinary skill in the art.Also, in certain applications it may be necessary or preferred topersons of ordinary skill in the art to use an amount outside theforegoing range.

In one embodiment, the method of using a compound as an anti-microbial,anti-cancer or anti-inflammatory involves administering an effectiveamount of -any of the compounds of Formulae I-V or compositions of thosecompounds. In a preferred embodiment, the method involves administeringthe compound represented by Formula II, to a patient in need of ananti-microbial, until the need is effectively reduced or more preferablyremoved.

As will be understood by one of skill in the art, “need” is not anabsolute term and merely implies that the patient can benefit from thetreatment of the anti-microbial, the anti-cancer, or anti-inflammatoryin use. By “patient” what is meant is an organism that can benefit bythe use of an anti-microbial, anti-cancer or anti-inflammatory agent.For example, any organism with B. anthracis, Plasmodium, Leishmania,Trypanosoma, and the like, may benefit from the application of ananti-microbial that may in turn reduce the amount of microbes present inthe patient. As another example, any organism with cancer, such as, acolorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, anon-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas,a melanoma, and the like, may benefit from the application of ananti-cancer agent that may in turn reduce the amount of cancer presentin the patient. Furthermore, any organism with an inflammatoryconditions, such as, rheumatoid arthritis, asthma, multiple sclerosis,psoriasis, stroke, myocardial infarction, and the like, may benefit fromthe application of an anti-inflammatory that may in turn reduce theamount of cells associated with the inflammatory response present in thepatient. In one embodiment, the patient's health may not require that ananti-microbial, anti-cancer, or anti-inflammatory be administered,however, the patient may still obtain some benefit by the reduction ofthe level of microbes, cancer cells, or inflammatory cells present inthe patient, and thus be in need. In one embodiment, the anti-microbialor anti-cancer agent is effective against one type of microbe or cancer,but not against other types; thus, allowing a high degree of selectivityin the treatment of the patient. In other embodiments, theanti-inflammatory may be effective against inflammatory conditionscharacterized by different cells associated with the inflammation. Inchoosing such an anti-microbial, anti-cancer or anti-inflammatory agent,the methods and results disclosed in the Examples may be useful. In analternative embodiment, the anti-microbial may be effective against abroad spectrum of microbes, preferably a broad spectrum of foreign, and,more preferably, harmful bacteria, to the host organism. In embodiments,the anti-cancer and/or anti-inflammatory agent may be effective againsta broad spectrum of cancers and inflammatoryconditions/cells/substances. In yet another embodiment, theanti-microbial is effective against all microbes, even those native tothe host. Examples of microbes that may be targets of anti-microbials,include, but are not limited to, B. anthracis, Plasmodium, Leishmania,Trypanosoma, and the like. In still further embodiments, the anti-canceragent is effective against a broad spectrum of cancers or all cancers.Examples of cancers, against which the compounds may be effectiveinclude a colorectal carcinoma, a prostate carcinoma, a breastadenocarcinoma, a non-small cell lung carcinoma, an ovarian carcinoma,multiple myelomas, a melanoma, and the like. Exemplary inflammatoryconditions against which the agents are effective include rheumatoidarthritis, asthma, multiple sclerosis, psoriasis, stroke, myocardialinfarction, and the like.

“Therapeutically effective amount,” “pharmaceutically effective amount,”or similar term, means that amount of drug or pharmaceutical agent thatwill result in a biological or medical response of a cell, tissue,system, animal, or human that is being sought. In a preferredembodiment, the medical response is one sought by a researcher,veterinarian, medical doctor, or other clinician.

“Anti-microbial” refers to a compound that reduces the likelihood ofsurvival of microbes, or blocks or alleviates the deleterious effects ofa microbe. In one embodiment, the likelihood of survival is determinedas a function of an individual microbe; thus, the anti-microbial willincrease the chance that an individual microbe will die. In oneembodiment, the likelihood of survival is determined as a function of apopulation of microbes; thus, the anti-microbial will increase thechances that there will be a decrease in the population of microbes. Inone embodiment, anti-microbial means antibiotic or other similar term.Such anti-microbials are capable of blocking the harmful effects,destroying or suppressing the growth or reproduction of microorganisms,such as bacteria. For example, such antibacterials and otheranti-microbials are described in Antibiotics, Chemotherapeutics andAntibacterial Agents for Disease Control (M. Grayson, editor, 1982), andE. Gale et al., The Molecular Basis of Antibiotic Action 2d edition(1981). In another embodiment, an anti-microbial will not change thelikelihood of survival, but will change the chances that the microbeswill be harmful to the host in some way. For instance, if the microbesecretes a substance that is harmful to the host, the anti-microbial mayact upon the microbe to stop the secretion or may counteract or blockthe harmful effect. In one embodiment, an anti-microbial, while,increasing the likelihood that the microbe(s) will die, is minimallyharmful to the surrounding, non-microbial, cells. In an alternativeembodiment, it is not important how harmful the anti-microbial is tosurrounding, nonmicrobial, cells, as long as it reduces the likelihoodof survival of the microbe.

“Anti-cancer agent” refers to a compound or composition including thecompound that reduces the likelihood of survival of a cancer cell. Inone embodiment, the likelihood of survival is determined as a functionof an individual cancer cell; thus, the anti-cancer agent will increasethe chance that an individual cancer cell will die. In one embodiment,the likelihood of survival is determined as a function of a populationof cancer cells; thus, the anti-cancer agent will increase the chancesthat there will be a decrease in the population of cancer cells. In oneembodiment, anti-cancer agent means chemotherapeutic agent or othersimilar term.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of a neoplastic disease, such as cancer. Examples ofchemotherapeutic agents include alkylating agents, such as a nitrogenmustard, an ethyleneimine and a methylmelamine, an alkyl sulfonate, anitrosourea, and a triazene, folic acid antagonists, anti-metabolites ofnucleic acid metabolism, antibiotics, pyrimidine analogs,5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids,triazol nucleosides, corticosteroids, a natural product such as a vincaalkaloid, an epipodophyllotoxin, an antibiotic, an enzyme, a taxane, anda biological response modifier; miscellaneous agents such as a platinumcoordination complex, an anthracenedione, an anthracycline, asubstituted urea, a methyl hydrazine derivative, or an adrenocorticalsuppressant; or a hormone or an antagonist such as anadrenocorticosteroid, a progestin, an estrogen, an antiestrogen, anandrogen, an antiandrogen, or a gouadotropin-releasing hormone analog.Specific examples include Adriamycin, Doxorubicin, 5-Fluorouracil,Cytosine arabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan,Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan,Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,Esperamicins, Melphalan, and other related nitrogen mustards. Alsoincluded in this definition are hormonal agents that act to regulate orinhibit hormone action on tumors, such as tamoxifen and onapristone.

The anti-cancer agent may act directly upon a cancer cell to kill thecell, induce death of the cell, to prevent division of the cell, and thelike. Alternatively, the anti-cancer agent may indirectly act upon thecancer cell by limiting nutrient or blood supply to the cell, forexample. Such anti-cancer agents are capable of destroying orsuppressing the growth or reproduction of cancer cells, such as acolorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, anon-small cell lung carcinoma, an ovarian carcinoma, multiple myelomas,a melanoma, and the like.

A “neoplastic disease” or a “neoplasm” refers to a cell or a populationof cells, including a tumor or tissue (including cell suspensions suchas bone marrow and fluids such as blood or serum), that exhibitsabnormal growth by cellular proliferation greater than normal tissue.Neoplasms can be benign or malignant.

An “inflammatory condition” includes, for example, conditions such asischemia, septic shock, autoimmune diseases, rheumatoid arthritis,inflammatory bowel disease, systemic lupus eythematosus, multiplesclerosis, asthma, osteoarthritis, osteoporosis, fibrotic diseases,dermatosis, including psoriasis, atopic dermatitis and ultravioletradiation (UV)-induced skin damage, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,Hodgkins disease, cachexia, inflammation associated with infection andcertain viral infections, including acquired immune deficiency syndrome(AIDS), adult respiratory distress syndrome and Ataxia Telangiestasia.

In one embodiment, a described compound, preferably a compound havingthe Formulae I-V, including those as described herein, is considered aneffective anti-microbial, anti-cancer, or anti-inflammatory if thecompound can influence 10% of the microbes, cancer cells, orinflammatory cells, for example. In a more preferred embodiment, thecompound is effective if it can influence 10 to 50% of the microbes,cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 50-80% of themicrobes, cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 80-95% of themicrobes, cancer cells, or inflammatory cells. In an even more preferredembodiment, the compound is effective if it can influence 95-99% of themicrobes, cancer cells, or inflammatory cells. “Influence” is defined bythe mechanism of action for each compound. Thus, for example, if acompound prevents the reproduction of microbes, then influence is ameasure of prevention of reproduction. Likewise, if a compound destroysmicrobes, then influence is a measure of microbe death. Also, forexample, if a compound prevents the division of cancer cells, theninfluence is a measure of prevention of cancer cell division. Further,for example, if a compound prevents the proliferation of inflammatorycells, then influence is a measure of prevention of inflammatory cellproliferation. Not all mechanisms of action need be at the samepercentage of effectiveness. In an alternative embodiment, a lowpercentage effectiveness may be desirable if the lower degree ofeffectiveness is offset by other factors, such as the specificity of thecompound, for example. Thus a compound that is only 10% effective, forexample, but displays little in the way of harmful side-effects to thehost, or non-harmful microbes or cells, can still be consideredeffective.

In one embodiment, the compounds described herein are administeredsimply to remove microbes, cancer cells or inflammatory cells, and neednot be administered to a patient. For example, in situations wheremicrobes can present a problem, such as in food products, the compoundsdescribed herein can be administered directly to the products to reducethe risk of microbes in the products. Alternatively, the compounds canbe used to reduce the level of microbes present in the surroundingenvironment, such working surfaces. As another example, the compoundscan be administered ex vivo to a cell sample, such as a bone marrow orstem cell transplant to ensure that only non-cancerous cells areintroduced into the recipient. After the compounds are administered theymay optionally be removed. This may be particularly desirable insituations where work surfaces or food products may come into contactwith other surfaces or organisms that could risk being harmed by thecompounds. In an alternative embodiment, the compounds may be left inthe food products or on the work surfaces to allow for a moreprotection. Whether or not this is an option will depend upon therelative needs of the situation and the risks associated with thecompound, which in part can be determined as described in the Examplesbelow.

The following non-limiting examples are meant to describe the preferredembodiments of the methods. Variations in the details of the particularmethods employed and in the precise chemical compositions obtained willundoubtedly be appreciated by those of skill in the art.

EXAMPLES Example 1 Fermentation of Compound of Formulae II-16 II-20. andII-24C

Strain CNB476 was grown in a 500-ml flask containing 100 ml ofvegetative medium consisting of the following per liter of deionizedwater: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; andsynthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The firstseed culture was incubated at 28 degree C. for 3 days on a rotary shakeroperating at 250 rpm. Four ml each of the first seed culture wasinoculated into three 500-ml flasks containing of 100 ml of thevegetative medium. The second seed cultures were incubated at 28 degreeC. and 250 rpm on a rotary shaker for 2 days. Four ml each of the secondseed culture was inoculated into thirty-five 500-ml flasks containing of100 ml of the vegetative medium. The third seed cultures were incubatedat 28 degree and 250 rpm on a rotary shaker for 2 days. Four ml each ofthe third seed culture was inoculated into four hundred 500-ml flaskscontaining 100 ml of the production medium consisting of the followingper liter of deionized water: starch, 10 g; yeast extract, 4 g; Hy-Soy,4 g; ferric sulfate, 40 mg; potassium bromide, 100 mg; calciumcarbonate, 1 g; and synthetic sea salt (Instant Ocean, AquariumSystems), 30 g. The production cultures were incubated at 28 degree C.and 250 rpm on roatry shakers for 1 day. Approximately 2 to 3 grams ofsterile Amberlite XAD-7 resin were added to the production cultures. Theproduction cultures were further incubated at 28 degree C. and 250 rpmon rotary shakers for 5 days. The culture broth was filtered throughcheese cloth to recover the Amberlite XAD-7 resin. The resin wasextracted with 2 times 6 liters ethyl acetate followed by 1 time 1.5liters ethyl acetate. The combined extracts were dried in vacuo. Thedried extract, containing 3.8 grams the compound of Formula II-16 andlesser quantities of compounds of formulae II-20 and II-24C, was thenprocessed for the recovery of the compounds of Formula II-16, II-20 andII-24C.

Example 2 Purification of Compound of Formulae II-16, II-20 and II-24C

The pure compounds of Formulae II-16, II-20 and II-24C were obtained byflash chromatography followed by HPLC. Eight grams crude extractcontaining 3.8 grams of the compound of Formula II-16 and lesserquantities of II-20 and II-24C was processed by flash chromatographyusing Biotage Flash40i system and Flash 40M cartridge (KP-Sil Silica,32-63 μm, 90 grams). The flash chromatography was developed by thefollowing step gradient:

-   -   1. Hexane (1 L)    -   2. 10% Ethyl acetate in hexane (1 L)    -   3. 20% Ethyl acetate in hexane, first elution (1 L)    -   4. 20% Ethyl acetate in hexane, second elution (1 L)    -   5. 20% Ethyl acetate in hexane, third elution (1 L)    -   6. 25% Ethyl acetate in hexane (1 L)    -   7. 50% Ethyl acetate in hexane (1 L)    -   8. Ethyl acetate (1 L)

Fractions containing the compound of Formula II-16 in greater or equalto 70% UV purity by HPLC were pooled and subject to HPLC purification,as described below, to obtain II-16, along with II-20 and II-24C, eachas pure compounds Column Phenomenex Luna 10u Silica Dimensions 25 cm ×21.2 mm ID Flow rate 25 ml/min Detection ELSD Solvent Gradient of 24%EtOAc/hexane for 19 min, 24% EtOAc/hexane to 100% EtOAc in 1 min, then100% EtOAc for 4 min

The fraction enriched in compound of Formula II-16 (described above;˜70% pure with respect to II-16) was dissolved in acetone (60 mg/ml).Aliquots (950 ul) of this solution were injected onto a normal-phaseHPLC column using the conditions described above. The compound ofFormula II-16 eluted at about 14 minutes, and minor compounds II-24C andII-20 eluted at 11 and 23 minutes, respectively. Fractions containingII-16, II-24C, and II-20 were pooled based on composition of compoundpresent. Fractions containing the desired compounds were concentratedunder reduced pressure to yield pure compound of Formula II-16, as wellas separate fractions containing II-24C and II-20, which were furtherpurified as described below.

Sample containing II-24C (70 mg) was dissolved in acetonitrile at aconcentration of 10 mg/ml, and 500 μl was loaded on an HPLC column ofdimensions 21 mm i.d. by 15 cm length containing Eclipse XDB-C18support. The solvent gradient increased linearly from 15%acetonitrile/85% water to 100% acetonitrile over 23 minutes at a flowrate of 14.5 ml/min. The solvent composition was held at 100%acetonitrile for 3 minutes before returning to the starting solventmixture. Compound II-24C eluted at 19 minutes as a pure compound underthese conditions.

To obtain pure compound II-20, the enriched samples generated from thepreparative HPLC method described above were triturated with EtOAc toremove minor lipophilic impurities. The resulting sample containedcompound II-20 in >95% purity.

Compound of Formula II-16: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. LowRes. Mass: m/z 314 (M+H), 336 (M+Na).

Compound of Formula II-20: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm. LowRes. Mass: m/z 266 (M+H). FIG. 7 depicts the 1 H NMR spectrum of acompound having the structure of Formula II-20.

Compound of Formula II-24C: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm.Low Res. Mass: m/z 328 (M+H), 350 (M+Na). FIG. 8 depicts the 1H NMRspectrum of a compound having the structure of Formula II-24C.

Example 3 Fermentation of Compounds of Formulae II-17 and II-18

Strain CNB476 was grown in a 500-ml flask containing 100 ml of the firstvegetative medium consisting of the following per liter of deionizedwater: glucose, 4 g; Bacto tryptone, 3 g; Bacto casitone, 5 g; andsynthetic sea salt (Instant Ocean, Aquarium Systems), 30 g. The firstseed culture was incubated at 28 degree C. for 3 days on a rotary shakeroperating at 250 rpm. Five ml of the first seed culture was inoculatedinto a 500-ml flask containing 100 ml of the second vegetative mediumconsisting of the following per liter of deionized water: starch, 10 g;yeast extract, 4 g; peptone, 2 g; ferric sulfate, 40 mg; potassiumbromide, 100 mg; calcium carbonate, 1 g; and sodium bromide, 30 g. Thesecond seed cultures were incubated at 28° C. for 7 days on a rotaryshaker operating at 250 rpm. Approximately 2 to 3 gram of sterileAmberlite XAD-7 resin were added to the second seed culture. The secondseed culture was further incubated at 28° C. for 2 days on a rotaryshaker operating at 250 rpm. Five ml of the second seed culture wasinoculated into a 500-ml flask containing 100 ml of the secondvegetative medium. The third seed culture was incubated at 28° C. for 1day on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gramof sterile Amberlite XAD-7 resin were added to the third seed culture.The third seed culture was further incubated at 28° C. for 2 days on arotary shaker operating at 250 rpm. Five ml of the third culture wasinoculated into a 500-ml flask containing 100 ml of the secondvegetative medium. The fourth seed culture was incubated at 28° C. for 1day on a rotary shaker operating at 250 rpm. Approximately 2 to 3 gramof sterile Amberlite XAD-7 resin were added to the fourth seed culture.The fourth seed culture was further incubated at 28° C. for 1 day on arotary shaker operating at 250 rpm. Five ml each of the fourth seedculture was inoculated into ten 500-ml flasks containing 100 ml of thesecond vegetative medium. The fifth seed cultures were incubated at 28°C. for 1 day on a rotary shaker operating at 250 rpm. Approximately 2 to3 grams of sterile Amberlite XAD-7 resin were added to the fifth seedcultures. The fifth seed cultures were further incubated at 28° C. for 3days on a rotary shaker operating at 250 rpm. Four ml each of the fifthseed culture was inoculated into one hundred and fifty 500-ml flaskscontaining 100 ml of the production medium having the same compositionas the second vegetative medium. Approximately 2 to 3 grams of sterileAmberlite XAD-7 resin were also added to the production culture. Theproduction cultures were incubated at 28° C. for 6 day on a rotaryshaker operating at 250 rpm. The culture broth was filtered throughcheese cloth to recover the Amberlite XAD-7 resin. The resin wasextracted with 2 times 3 liters ethyl acetate followed by 1 time 1 literethyl acetate. The combined extracts were dried in vacuo. The driedextract, containing 0.42 g of the compound Formula II-17 and 0.16 gramthe compound of Formula II-18, was then processed for the recovery ofthe compounds.

Example 4 Purification of Compounds of Formula II-17 and II-18

The pure compounds of Formula II-17 and II-18 were obtained byreversed-phase HPLC as described below: Column ACE 5 C18-HL Dimensions15 cm × 21 mm ID Flow rate 14.5 ml/min Detection 214 nm Solvent Gradientof 35% Acetonitrile/65% H₂O to 90% Acetonitrile/10% H₂O over 15 min

Crude extract (100 mg) was dissolved in 15 ml of acetonitrile. Aliquots(900 ul) of this solution were injected onto a reversed-phase HPLCcolumn using the conditions described above. Compounds of Formulae II-17and II-18 eluted at 7.5 and 9 minutes, respectively. Fractionscontaining the pure compounds were first concentrated using nitrogen toremove organic solvent. The remaining solution was then frozen andlyophilized to dryness.

Compound of Formula II-17: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm.High Res. Mass (APCI): m/z 280.156 (M+H), Δ_(calc)=2.2 ppm, C₁₅H₂₂NO₄FIG. 49 depicts the ¹H NMR spectrum of a compound having the structureof Formula II-17.

Compound of Formula II-18: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm.High Res. Mass (APCI): m/z 358.065 (M+H), Δ_(calc)=−1.9 ppm,C₁₅H₂₁NO₄Br. FIG. 50 depicts the ¹H NMR spectrum of a compound havingthe structure of Formula II-18.

Example 5 Preparation of Compound of Formula II-19 from II-16

A sample of compound of Formula II-16 (250 mg) was added to an acetonesolution of sodium iodide (1.5 g in 10 ml) and the resulting mixturestirred for 6 days. The solution was then filtered through a 0.45 micronsyringe filter and injected directly on a normal phase silica HPLCcolumn (Phenomenex Luna 10 u Silica, 25 cm×21.2 mm) in 0.95 ml aliquots.The HPLC conditions for the separation of compound formula II-19 fromunreacted II-16 employed an isocratic HPLC method consisting of 24%ethyl acetate and 76% hexane, in which the majority of compound II-19eluted 2.5 minutes before compound II-16. Equivalent fractions from eachof 10 injections were pooled to yield 35 mg compound II-19. CompoundII-19: UV (Acetonitrile/H₂O) 225 (sh), 255 (sh) nm; ESMS, m/z 406.0(M+H); ¹H NMR in DMSO-d₆ (see FIG. 9).

Example 6 Synthesis of the Compounds of Formulae II-2, II-3, and II-4

Compounds of Formulae II-2, II-3 and II-4 can be synthesized fromcompounds of Formulae II-16, II-17 and II-18, respectively, by catalytichydrogenation.Exemplary Depiction of Synthesis

Example 6A Catalytic Hydrogenation of Compound of Formula II-16

Compound of Formula II-16 (10 mg) was dissolved in acetone (5 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (1-2mg)and a magnetic stirrer bar. The reaction mixture was stirred in ahydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 3 cc silica column and washed withacetone. The filtrate was filtered again through 0.2 μm Gelman Acrodiscto remove any traces of catalyst. The solvent was evaporated off fromfiltrate under reduced pressure to yield the compound of Formula II-2 asa pure white powder: UV (acetonitrile/H₂O): λ_(max) 225 (sh) nm. FIG. 10depicts the NMR spectrum of the compound of Formula II-2 in DMSO-d6.FIG. 11 depicts the low resolution mass spectrum of the compound ofFormula II-2: m/z 316 (M+H), 338 (M+Na).

Example 6B Catalytic Hydrogenation of Compound of Formula II-17

Compound of Formula II-17 (5 mg) was dissolved in acetone (3 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (aboutlmg) and a magnetic stirrer bar. The reaction mixture was stirred in ahydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 0.2 μm Gelman Acrodisc to remove thecatalyst. The solvent was evaporated off from filtrate to yield thecompound of Formula II-3 as a white powder which was purified by normalphase HPLC using the following conditions: Column: Phenomenex Luna 10uSilica Dimensions: 25 cm × 21.2 mm ID Flow rate: 14.5 ml/min Detection:ELSD Solvent: 5% to 60% EtOAc/Hex for 19 min, 60 to 100% EtOAc in 1 min,then 4 min at 100% EtOAc

Compound of Formula II-3 eluted at 22.5 min as a pure compound: UV(acetonitrile/H₂O): λ_(max) 225 (sh) nm. FIG. 12 depicts the NMRspectrum of the compound of Formula II-3 in DMSO-d6. FIG. 13 depicts thelow resolution mass spectrum of the compound of Formula II-3: m/z 282(M+H), 304 (M+Na).

Example 6C Catalytic Hydrogenation of Compound of Formula II-18

3.2 mg of compound of Formula II-18 was dissolved in acetone (3 mL) in ascintillation vial (20 mL) to which was added the 10% (w/w) Pd/C (about1 mg) and a magnetic stirrer bar. The reaction mixture was stirred inhydrogen atmosphere at room temperature for about 15 hours. The reactionmixture was filtered through a 0.2 μm Gelman Acrodisc to remove thecatalyst. The solvent was evaporated off from filtrate to yield thecompound of Formula II-4 as a white powder which was further purified bynormal phase HPLC using the following conditions: Column: PhenomenexLuna 10u Silica Dimensions: 25 cm × 21.2 mm ID Flow rate: 14.5 ml/minDetection: ELSD Solvent: 5% to 80% EtOAc/Hex for 19 min, 80 to 100%EtOAc in 1 min, then 4 min at 100% EtOAc

Compound of Formula II-4 eluted at 16.5 min as a pure compound: UV(acetonitrile/H₂O): λ_(max) 225 (sh) nm. FIG. 14 depicts the NMRspectrum of the compound of Formula II-4 in DMSO-d6. FIG. 15 depicts thelow resolution mass of the compound of Formula II-4: m/z 360 (M+H), 382(M+Na).

Example 7 Synthesis of the Compounds of Formulae II-5A and II-5B

Compounds of Formula II-5A and Formula II-5B can be synthesized fromcompound of Formula II-16 by epoxidation with mCPBA.

Compound of Formula II-16 (101 mg, 0.32 mmole) was dissolved inmethylenechloride (30 mL) in a 100 ml of round bottom flask to which wasadded 79 mg (0.46 mmole) of meta-chloroperbenzoic acid (mCPBA) and amagnetic stir bar. The reaction mixture was stirred at room temperaturefor about 18 hours. The reaction mixture was poured onto a 20 cc silicaflash column and eluted with 120 ml of CH₂Cl₂, 75 ml of 1:1 ethylacetate/hexane and finally with 40 ml of 100% ethyl acetate. The 1:1ethyl acatete/hexane fractions yield a mixture of diastereomers ofepoxyderivatives, Formula II-5A and II-5B, which were separated bynormal phase HPLC using the following conditions: Column Phenomenex Luna10u Silica Dimensions 25 cm × 21.2 mm ID Flow rate 14.5 ml/min DetectionELSD Solvent 25% to 80% EtOAc/Hex over 19 min, 80 to 100% EtOAc in 1min, then 5 min at 100% EtOAc

Compound Formula II-5A (major product) and II-5B (minor product) elutedat 21.5 and 19 min, respectively, as pure compounds. Compound II-5B wasfurther chromatographed on a 3cc silica flash column to remove traces ofchlorobenzoic acid reagent.

Structural Characterization

Formula II-5A: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm. Low Res. Mass:m/z 330 (M+H), 352 (M+Na). FIGS. 16-17, respectively depict the 1H NMRspectrum of Formula II-5A and the mass spectrum of Formula II-5A.

Formula II-5B: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm. Low Res. Mass:m/z 330 (M+H), 352 (M+Na). FIGS. 18-19, respectively depict the 1H NMRspectrum of II-5B and the mass spectrum of II-5B.

Example 8 Synthesis of the Compounds of Formulae IV-1 IV-2, IV-3 andIV-4 Synthesis of Diol Derivatives (Formula IV-2)

Diols may be synthesized by Sharpless dihydroxylation using AD mix-α andβ: AD mix-α is a premix of four reagents, K₂OsO₂(OH)₄; K₂CO₃; K₃Fe(CN)₆;(DHQ)₂-PHAL [1,4-bis(9-O-dihydroquinine)phthalazine] and AD mix-β is apremix of K₂OsO₂(OH)₄; K₂CO₃; K₃Fe(CN)₆; (DHQD)₂-PHAL[1,4-bis(9-O-dihydroquinidine)phthalazine] which are commerciallyavailable from Aldrich. Diol can also be synthesized by acid or basehydrolysis of epoxy compounds (Formula II-5A and II-5B) which may bedifferent to that of products obtained in Sharpless dihydroxylation intheir stereochemistry at carbons bearing hydroxyl groups

Sharpless Dihydroxylation of Compounds II-16 II-17 and II-18

Any of the compounds of Formulae II-16, II-17 and II-18 may be used asthe starting compound. In the example below, compound of Formula II-16is used. The starting compound is dissolved in t-butanol/water in around bottom flask to which is added AD mix-α or β and a magnetic stirbar. The reaction is monitored by silica TLC as well as massspectrometer. The pure diols are obtained by usual workup andpurification by flash chromatography or HPLC. The structures areconfirmed by NMR spectroscopy and mass spectrometry. In this method bothhydroxyl groups are on same side.

Nucleophilic Ring Opening of Epoxy Compounds (11-5):

The epoxy ring is opened with various nucleophiles like NaCN, NaN₃,NaOAc, HBr, HCl, etc. to creat various substituents on the cyclohexanering, including a hydroxyl substituent.Examples:

The epoxy is opened with HCl to make Formula IV-3:

Compound of Formula II-5A (3.3 mg) was dissolved in acetonitrile (0.5ml) in a 1 dram vial to which was added 5% HCl (500 ul) and a magneticstir bar. The reaction mixture was stirred at room temperature for aboutan hour. The reaction was monitored by mass spectrometry. The reactionmixture was directly injected on normal phase HPLC to obtain compound ofFormula IV-3C as a pure compound without any work up. The HPLCconditions used for the purification were as follows: Phenomenex Luna 10u Silica column (25 cm×21.2 mm ID) with a solvent gradient of 25% to 80%EtOAc/Hex over 19 min, 80 to 100% EtOAc in 1 min, then 5 min at 100%EtOAc at a flow rate of 14.5 ml/min. An ELSD was used to monitor thepurification process. Compound of Formula IV-3C eluted at about 18 min(2.2 mg). Compound of Formula IV-3C: UV (Acetonitrile/H₂O) λ_(max) 225(sh) nm; ESMS, m/z 366 (M+H), 388 (M+Na); ¹H NMR in DMSO-d₆ (FIG. 20)The stereochemistry of the compound of Formula IV-3C was determinedbased on coupling constants observed in the cyclohexane ring in 1:1C₆D₆/DMSO-d₆ (FIG. 21)

Reductive ring opening of epoxides (II-5): The compound of Formula istreated with metalhydrides like BH₃-THF complex to make compound ofFormula IV-4.

Example 9 Synthesis of the Compounds of Formulae II-13C and II-8C

Compound of Formula II-16 (30 mg) was dissolved in CH₂Cl₂ (6 ml) in ascintillation vial (20 ml) to which Dess-Martin Periodinane (122 mg) anda magnetic stir bar were added. The reaction mixture was stirred at roomtemperature for about 2 hours. The progress of the reaction wasmonitored by TLC (Hex:EtOAc, 6:4) and analytical HPLC. From the reactionmixture, the solvent volume was reduced to one third, absorbed on silicagel, poured on top of a 20 cc silica flash column and eluted in 20 mlfractions using a gradient of Hexane/EtOAc from 10 to 100%. The fractioneluted with 30% EtOAc in Hexane contained a mixture of rotamers ofFormula II-13C in a ratio of 1.5:8.5. The mixture was further purifiedby normal phase HPLC using the Phenomenex Luna 10 u Silica column (25cm×21.2 mm ID) with a solvent gradient of 25% to 80% EtOAc/Hex over 19min, 80 to 100% EtOAc over 1 min, holding at 100% EtOAc for 5 min, at aflow rate of 14.5 ml/min. An ELSD was used to monitor the purificationprocess. Compound of Formula II-13C eluted at 13.0 and 13.2 mins as amixture of rotamers with in a ratio of 1.5:8.5 (7 mg). Formula II-13C:UV (Acetonitrile/H₂O) λ_(max) 226 (sh) & 300 (sh) nm; ESMS, m/z 312(M+H)⁺, 334 (M+Na)⁺; ¹H NMR in DMSO-d₆ (see FIG. 22).

The rotamer mixture of Formula II-13C (4 mg) was dissolved in acetone (1ml) in a scintillation vial (20 ml) to which a catalytic amount (0.5 mg)of 10% (w/w) Pd/C and a magnetic stir bar were added. The reactionmixture was stirred in a hydrogen atmosphere at room temperature forabout 15 hours. The reaction mixture was filtered through a 0.2 μmGelman Acrodisc to remove the catalyst. The solvent was evaporated fromthe filtrate to yield compound of Formula II-8C as a colorless gum whichwas further purified by normal phase HPLC using a Phenomenex Luna 10 uSilica column (25 cm×21.2 mm ID) with a solvent gradient of 25% to 80%EtOAc/Hex over 19 min, 80 to 100% EtOAc over 1 min, holding at 100%EtOAc for 5 min, at a flow rate of 14.5 ml/min. An ELSD was used tomonitor the purification process. Compound of Formula II-8C (1 mg)eluted at 13.5 min as a pure compound. Formula II-8C: UV(Acetonitrile/H₂O) λ_(max) 225 (sh) nm; ESMS, m/z 314 (M+H)⁺, 336(M+Na)⁺; ¹H NMR in DMSO-d₆ (See FIG. 23).

Example 10 Synthesis of the Compound of Formulae II-25 from II-13C

The rotamer mixture of Formula II-13C (5 mg) was dissolved in dimethoxyethane (monoglyme; 1.5 ml) in a scintillation vial (20 ml) to whichwater (15 μl (1% of the final solution concentration)) and a magneticstir bar were added. The above solution was cooled to −78° C. on a dryice-acetone bath, and a sodium borohydride solution (3.7 mg of NaBH₄ in0.5 ml of monoglyme (created to allow for slow addition)) was addeddrop-wise. The reaction mixture was stirred at −78° C. for about 14minutes. The reaction mixture was acidified using 2 ml of 4% HClsolution in water and extracted with CH₂Cl₂. The organic layer wasevaporated to yield mixture of compound of formulae II-25 and II-16 in a9.5:0.5 ratio as a white solid, which was further purified by normalphase HPLC using a Phenomenex Luna 10 u Silica column (25 cm×21.2 mmID). The mobile phase was 24% EtOAc/76% Hexane, which was held isocraticfor 19 min, followed by a linear gradient of 24% to 100% EtOAc over 1min, and held at 100% EtOAc for 3 min; the flow rate was 25 ml/min. AnELSD was used to monitor the purification process. Compound of formulaII-25 (1.5 mg) eluted at 11.64 min as a pure compound. Compound ofFormula II-25: UV (Acetonitrile/H₂O) λ_(max) 225 (sh) nm; ESMS, m/z 314(M+H)⁺, 336 (M+Na)⁺; ¹H NMR in DMSO-d₆ (see FIG. 24).

Example 11

Synthesis of the Compound of Formulae II-21 from II-19

Acetone (7.5 ml) was vigorously mixed with 5 N NaOH (3 ml) and theresulting mixture evaporated to a minimum volume in vacuo. A sample of100 l of this solution was mixed with compound of Formula II-19 (6.2 mg)in acetone (1 ml) and the resulting biphasic mixture vortexed for 2minutes. The reaction solution was immediately subjected to preparativeC18 HPLC. Conditions for the purification involved a linear gradient if10% acetonitrile/90% water to 90% acetonitrile/ 10% water over 17minutes using an Ace 5μ C18 HPLC column of dimensions 22 mm id by 150 mmlength. Compound of Formula II-21 eluted at 9.1 minutes under theseconditions to yield 0.55 mg compound. Compound of Formula II-21: UV(Acetonitrile/H₂O) 225 (sh), ESMS, m/z 296.1 (M+H); ¹H NMR in DMSO-d₆(see FIG. 25).

Example 12 Synthesis of the Compound of Formulae II-22 from II-19

A sample of 60 mg sodium propionate was added to a solution of compoundof Formula II-19 (5.3 mg) in DMSO (1 ml) and the mixture sonicated for 5minutes, though the sodium propionate did not completely dissolve. After45 minutes, the solution was filtered through a 0.45 [ syringe filterand purified directly using HPLC. Conditions for the purificationinvolved a linear gradient if 10% acetonitrile/90% water to 90%acetonitrile/ 10% water over 17 minutes using an Ace 5μ C18 HPLC columnof dimensions 22 mm id by 150 mm length. Under these conditions,compound of Formula II-22 eluted at 12.3 minutes to yield 0.7 mgcompound (15% isolated yield). UV (Acetonitrile/H₂O) 225 (sh), ESMS, m/z352.2 (M+H); ¹H NMR in DMSO-d₆ (see FIG. 26).

Example 13 Oxidation of Secondary Hydroxyl Group in Compounds ofFormulae II-16, II-17 and II-18 and Reaction with Hydroxy or MethoxyAmines

Any of the compounds of Formulae II-16, II-17 and II-18 may be used asthe starting compound. The secondary hydroxyl group in the startingcompound is oxidized using either of the following reagents: pyridiniumdichromate (PDC), pyridinium chlorochromate (PCC), Dess-Martinperiodinane or oxalyl chloride (Swern oxidation) (Ref: OrganicSyntheses, collective volumes I-VIII). Preferably, Dess-Martinperiodinane may be used as a reagent for this reaction. (Ref: FenteanyG. et al. Science, 1995, 268, 726-73). The resulting keto compound istreated with hydroxylamine or methoxy amine to generate oximes.

Examples

Example 14 Reductive Amination of Keto-Derivative

The keto derivatives, for example Formula II-8 and II-13, are treatedwith sodium cyanoborohydride (NaBH₃CN) in the presence of various basesto yield amine derivatives of the starting compounds which aresubsequently hydrogenated with 10% Pd/C, H₂ to reduce the double bond inthe cyclohexene ring.

Example

Example 15 Cyclohexene Ring Opening

Any compound of Formulae II-16, II-17 and II-18 may be used as astarting compound. The starting compound is treated with OsO₄ and NaIO₄in THF-H₂O solution to yield dial derivatives which are reduced toalcohol with NaBH₄ in the same pot.

Example

Example 16 Dehydration of Alcohol Followed by Aldehyde Formation atLactone-Lactam Ring Junction

A starting compound of any of Formulae II-16, II-17 or 11-18 is treatedwith mesylchloride in the presence of base to yield a dehydratedderivative. The resulting dehydrated compound is treated with OsO₄ andNaIO₄ in THF-H₂O to yield an aldehyde group at the lactone-lactam ringjunction.

Example 17 Various Reactions on Aldehyde Derivatives I-1

Wittig reactions are performed on the aldehyde group using variousphosphorus ylides [e.g., (triphenylphosphoranylidene)ethane] to yield anolefin. The double bond in the side chain is reduced by catalytichydrogenation.

Example

Reductive amination is performed on the aldehyde group using variousbases (eg. NH₃) and sodium cyanoborohydride to yield amine derivatives.Alternatively, the aldehyde is reduced with NaBH₄ to form alcohols inthe side chain.

Example

Organometallic addition reactions to the aldehyde carbonyl, such asGrignard reactions, may be performed using various alkyl magnesiumbromide or chloride reagents (eg. isopropylmagnesium bromide,phenylmagnesium bromide) to yield various substituted secondaryalcohols.

Example

Example 18 In Vitro Biology

Initial studies of a compound of Formula II-16, which is also referredto as Salinosporamide A, employed the National Cancer Institute (NCI)screening panel, which consists of 60 human tumor cell lines thatrepresent leukemia, melanoma and cancers of the lung, colon, brain,ovary, breast, prostate and kidney. A detailed description of thescreening procedure can be found at hypertext transfer protocol(http://) “dtp.nci.nih.gov/branches/btb/ivclsp.html.”

In brief, each of the 60 human tumor cell lines were grown in RPMI 1640medium, supplemented with 5% fetal bovine serum and 2 mM L-glutamine.Cells were plated at their appropriate density in 96-well microtiterplates and incubated at 37° C., 5% CO₂, 95% air and 100% relativehumidity. After 24 hours, 100 pL of various 10-fold serial dilutions ofSalinosporamide A were added to the appropriate wells containing 100 μLof cells, resulting in a final Salinosporamide A concentration rangingfrom 10 nM to 100 μM. Cells were incubated for an additional 48 hoursand a sulforhodamine B protein assay was used to estimate cell viabilityor growth.

Three dose response parameters were calculated as follows:

-   -   GI₅₀ indicates the concentration that inhibits growth by 50%.    -   TGI indicates the concentration that completely inhibits growth.    -   LC₅₀ indicates the concentration that is lethal to 50% of the        cells.

An example of a study evaluating Salinosporamide A in the NCI screen isshown in Table 1 below.

Data indicate that the mean GI₅₀ value of Salinosporamide A was lessthan 10 nM. The wide range (>1000-fold difference) observed in both themean TGI and mean LC₅₀ values for the most sensitive and the mostresistant tumor cell lines illustrates that Salinosporamide A displaysgood selectivity and does not appear to be a general toxin. Furthermore,the mean TGI data suggest that Salinosporamide A shows preferredspecificity towards melanoma and breast cancer cell lines. The assay wasrepeated and showed similar results.

The results of the NCI tumor screen show that Salinosporamide A: (1) isa potent compound with a mean GI₅₀ value of <10 nM, and (2) displaysgood tumor selectivity of more than 1000-fold difference in both themean TGI and mean LC₅₀ values between the most sensitive and resistanttumor cell lines.

Example 19 Growth Inhibition of Tumor Cell Lines

B16-F10 (ATCC; CRL-6475), DU 145 (ATCC; HTB-81), HEK293 (ATCC;CRL-1573), HT-29 (ATCC; HTB-38), LoVo (ATCC; CCL-229), MDA-MB-231 (ATCC;HTB-26), MIA PaCa-2 (ATCC; CRL-1420), NCI-H292 (ATCC; CRL-1848), OVCAR-3(ATCC, HTB-161), PANC-1 (ATCC; CRL-1469), PC-3 (ATCC; CRL-1435), RPMI8226 (ATCC; CCL-155) and U266 (ATCC; TIB-196) were maintained inappropriate culture media. The cells were cultured in an incubator at 37° C. in 5% CO2 and 95% humidified air.

For cell growth inhibition assays, B16-F10, DU 145, HEK293, HT-29, LoVo,MDA-MB-231, MIA PaCa-2, NCI-H292, OVCAR-3, PANC-1, PC-3, RPMI 8226 andU266 cells were seeded at 1.25×10³, 5×10³, 1.5×10⁴, 5×10³, 5×10³, 1×10⁴,2×10³, 4×10³, 1×10⁴, 7.5×10³, 5×10³, 2×10⁴, 2.5×10⁴ cells/wellrespectively in 90 μl complete media into Corning 3904 black-walled,clear-bottom tissue culture plates. 20 mM stock solutions of FormulaII-16 were prepared in 100% DMSO, aliquoted and stored at −80° C.Formula II-16 was serially diluted and added in triplicate to the testwells resulting in final concentrations ranging from of 20 μM to 0.2 μM.The plates were returned to the incubator for 48 hours. The finalconcentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (generated byXLfit 3.0, ID Business Solutions Ltd or Prism 3.0, GraphPad SoftwareInc).

The data in Table 2 summarize the growth inhibitory effects of FormulaII-16 against 13 diverse human and mouse tumor cell lines. TABLE 2 MeanEC₅₀ values of Formula II-16 against various tumor cell lines EC₅₀ (nM),Cell line Source mean ± SD* n B16-F10 Mouse, melanoma 47 ± 20 12 DU 145Human, prostate carcinoma 37 ± 10 3 HEK293 Human, embryonic kidney 47 2HT-29 Human, colorectal adenocarcinoma 40 ± 26 5 LoVo Human, colorectaladenocarcinoma 70 ± 8  3 MDA-MB-231 Human, breast adenocarcinoma 87 ± 4012 MIA PaCa-2 Human, pancreatic carcinoma 46 2 NCI-H292 Human, non smallcell lung 66 ± 29 12 carcinoma OVCAR-3 Human, ovarian adenocarcinoma 49± 31 6 PANC-1 Human, pancreatic carcinoma 60 2 PC-3 Human, prostateadenocarcinoma 64 ± 26 19 RPMI 8226 Human, multiple myeloma 8.6 ± 1.9 26U266 Human, multiple myeloma 4.7 ± 0.7 6*Where n (number of independent experiments) = 2, the mean value ispresented

The EC₅₀ values indicate that Formula II-16 was cytotoxic againstB16-F10, DU 145, HEK293, HT-29, LoVo, MDA-MB-231, MIA PaCa-2, NCI-H292,OVCAR-3, PANC-1, PC-3, RPMI 8226 and U266 cells.

Example 20 In vitro Inhibition of Proteasome Activity by Formulae II-2,II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,II-20, II-21, II-22, II-24C, II-25 and IV-3C

All the compounds were prepared as 20 mM stock solution in DMSO andstored in small aliquots at −80° C. Purified rabbit muscle 20Sproteasome was obtained from CalBiochem. To enhance thechymotrypsin-like activity of the proteasome, the assay buffer (20 mMHEPES, pH7.3, 0.5 mM EDTA, and 0.05% Triton X100) was supplemented withSDS resulting in a final SDS concentration of 0.035%. The substrate usedwas suc-LLVY-AMC, a fluorogenic peptide substrate specifically cleavedby the chymotrypsin-like activity of the proteasome. Assays wereperformed at a proteasome concentration of 1 μg/ml in a final volume of200 μl in 96-well Costar microtiter plates. Formula II-2, Formula II-4,Formula II-16, Formula II-17, Formula II-18, Formula II-19, FormulaII-21 and Formula II-22 were tested as eight-point dose response curveswith final concentrations ranging from 500 nM to 158 pM. Formula II-5A,Formula II-5B and Formula II-20 were tested at concentrations rangingfrom 1 μM to 0.32 nM. Formula II-3 was tested as an eight-dose responsecurve with final concentrations ranging from 10 μM to 3.2 nM, whileFormula II-8C, Formula II-13C, Formula II-24C, Formula II-25 and FormulaIV-3C were tested with final concentrations ranging from 20 μM to 6.3nM.The samples were incubated at 37° C. for five minutes in a temperaturecontrolled plate reader. During the preincubation step, the substratewas diluted 25-fold in SDS-containing assay buffer. After thepreincubation period, the reactions were initiated by the addition of 10μl of the diluted substrate and the plates were returned to the platereader. The final concentration of substrate in the reactions was 20 μM.All data were collected every five minutes for more than 1.5 hour andplotted as the mean of triplicate data points. The EC₅₀ values (the drugconcentration at which 50% of the maximal relative fluorescence unit isinhibited) were calculated by Prism (GraphPad Software) using asigmoidal dose-response, variable slope model. To evaluate the activityof the compounds against the caspase-like activity of the 20Sproteasomes, reactions were performed as described above except thatZ-LLE-AMC was used as the peptide substrate. Formulae II-3, II-4, II-5A,II-5B, II-8C, II-13C, II-17, II-18, II-20, II-21. II-22, II-24C, II-25and Formula IV-3C were tested at concentrations ranging from 20 μM to6.3 nM. Formula II-2 was tested at concentrations ranging from 10 μM to3.2 nM, while Formula II-16 and Formula II-19 were tested atconcentrations ranging from 5 μM to 1.58 nM. For the evaluation of thecompounds against the trypsin-like activity of the proteasome, the SDSwas omitted from the assay buffer and Boc-LRR-AMC was used as thepeptide substrate. Formula II-20 was tested at concentrations rangingfrom 5 μM to 1.6 nM. Formulae II-3, II-8C, II-13C, II-17, II-21, II-22,II-24C, II-25 and IV-3C were tested at concentrations ranging from 20 μMto 6.3 nM. For Formulae II-2 and II-5B the concentrations tested rangedfrom 10 μM to 3.2 nM, while Formulae II-4, II-5A, II-16, II-18 and II-19were tested at concentrations ranging from 1 μM to 0.32 nM.

Results (mean EC₅₀ values) are shown in Table 3 and illustrate thatamong the tested compounds, Formulae II-5A, II-16, II-18, II-19, II-20,II-21 and II-22 are the most potent inhibitors of the chymotrypsin-likeactivity of the 20S proteasome with EC₅₀ values ranging from 2.2 nM to 7nM. Formulae II-2, II-4, II-5B and II-17 inhibit the proteasomalchymotrypsin-like activity with EC₅₀ values ranging from 14.2 nM to 87nM, while the EC₅₀ value of Formula II-3 is 927 nM. Formula II-24C,II-13C and IV-3C inhibited the chymotrypsin-like activity with EC₅₀values of 2.2 μM, 8.2 μM and 7.8 μM respectively. EC₅₀ values forFormulae II-8C and II-25 were greater than 20 μM. Under the conditionstested, Formulae II-2, II-3, II-4, II-5A, II-5B, II-13C, II-16, II-17,II-18, II-19, II-20, II-21, II-22 and II-24C were able to inhibit thetrypsin-like activity of the 20S proteasome. Formulae II-4, II-5A,II-16, II-18 and II-19 inhibited the caspase-like activity with EC₅₀values ranging from 250 nM to 744 nM, while Formulae II-2, II-5B, II-17,II-20, II-21, and II-22 had EC₅₀ values ranging from 1.2 μM to 3.3 μM.TABLE 3 Effects of Formulae II-2, II-3, II-4, II-5A, II-5B, II-8C,II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-24C, II-25and IV-3C on the various enzymatic activities of purified rabbit 20Sproteasomes EC₅₀ Values Analog Chymotrypsin-like Trypsin-likeCaspase-like Formula II-2 18 nM 230 nM 1.5 μM Formula II-3 927 nM 6.6μM >20 μM Formula II-4 14.2 nM 109 nM 744 nM Formula II-5A 6.5 nM 89 nM487 nM Formula II-5B 87 nM 739 nM 3.3 μM Formula II-8C* >20 μM >20μM >20 μM Formula II-13C 8.2 μM 10.7 μM >20 μM Formula II-16 2.5 nM 21nM 401 nM Formula II-17 29.5 nM 588 nM 1.2 μM Formula II-18 2.2 nM 14 nM250 nM Formula II-19* 3 nM 13 nM 573 nM Formula II-20* 5 nM 318 nM 1.4μM Formula II-21* 7 nM 720 nM 2.6 μM Formula II-22* 5 nM 308 nM 1.3 μMFormula II-24C* 2.2 μM 3.2 μM >20 μM Formula II-25* >20 μM >20 μM >20 μMFormula IV-3C 7.8 μM >20 μM >20 μM*n = 1

Example 21 Salinosporamide A (II-16) Inhibits Chymotrypsin-Like Activityof Rabbit Muscle 20S Proteasomes

The effect of Salinosporamide A (11-16) on proteasomes was examinedusing a commercially available kit from Calbiochem (catalog no. 539158),which uses a fluorogenic peptide substrate to measure the activity ofrabbit muscle 20S proteasomes (Calbiochem 20S Proteasome Kit). Thispeptide substrate is specific for the chymotrypsin-like enzyme activityof the proteasome.

Omuralide was prepared as a 10 mM stock in DMSO and stored in 5 μLaliquots at −80° C. Salinosporamide A was prepared as a 25.5 mM solutionin DMSO and stored in aliquots at −80° C. The assay measures thehydrolysis of Suc-LLVY-AMC into Suc-LLVY and AMC. The released coumarin(AMC) was measured fluorometrically by using λ_(ex)=390 nm andλ_(em)=460 nm. The assays were performed in a microtiter plate (Corning3904), and followed kinetically with measurements every five minutes.The instrument used was a Thermo Lab Systems Fluoroskan, with theincubation chamber set to 37° C. The assays were performed according tothe manufacturer's protocol, with the following changes. The proteasomewas activated as described with SDS, and held on ice prior to the assay.Salinosporamide A and Omuralide were serially diluted in assay buffer tomake an 8-point dose-response curve. Ten microliters of each dose wereadded in triplicate to the assay plate, and 190 μL of the activatedproteasome was added and mixed. The samples were pre-incubated in theFluoroskan for 5 minutes at 37° C. Substrate was added and the kineticsof AMC were followed for one hour. All data were collected and plottedas the mean of triplicate data points. The data were normalized toreactions performed in the absence of Salinosporamide A and modeled inPrism as a sigmoidal dose-response, variable slope.

Similar to the results obtained for the in vitro cytotoxicity (Table 2),Feling, et al., Angew Chem Int Ed Engl 42:355 (2003), the EC₅₀ values inthe 20S proteasome assay showed that Salinosporamide A was approximately40-fold more potent than Omuralide, with an average value of 1.3 nMversus 49 nM, respectively (FIG. 27). This experiment was repeated andthe average EC₅₀ in the two assays was determined to be 2 nM forSalinosporamide A and 52 nM for Omuralide.

Salinosporamide A is a potent inhibitor of the chymotrypsin-likeactivity of the proteasome. The EC₅₀ values for cytotoxicity were in the10-200 nM range suggesting that the ability of Salinosporamide A toinduce cell death was due, at least in large part, to proteasomeinhibition. The data suggest that Salinosporamide A is a potent smallmolecule inhibitor of the proteasome.

Example 22 Salinosporamide A (II- 16) Inhibition of PGPH Activity ofRabbit Muscle 20S Proteasomes

Omuralide can inhibit the PGPH activity (also known as the caspase-like)of the proteasome; therefore, the ability of Salinosporamide A toinhibit the PGPH activity of purified rabbit muscle 20S proteasomes wasassessed. A commercially available fluorogenic substrate specific forthe PGPH activity was used instead of the chymotrypsin substratesupplied in the proteasome assay kit described above.

Salinosporamide A (II-16) was prepared as a 20 mM solution in DMSO andstored in small aliquots at −80° C. The substrate Z-LLE-AMC was preparedas a 20 mM stock solution in DMSO, stored at −20° C. The source of theproteasomes was the commercially available kit from Calbiochem (Cat.#539158). As with the chymotrypsin substrate, the proteasome can cleaveZ-LLE-AMC into Z-LLE and free AMC. The activity can then be determinedby measuring the fluorescence of the released AMC (λ_(ex)=390 nm andλ_(em)=460 nm). The proteasomes were activated with SDS and held on iceas per manufacturer's recommendation. Salinosporamide A was diluted inDMSO to generate a 400-fold concentrated 8-point dilution series. Theseries was diluted 20-fold with assay buffer and preincubated with theproteasomes as described for the chymotrypsin-like activity. Afteraddition of substrate, the samples were incubated at 37° C., and releaseof the fluorescent AMC was monitored in a fluorimeter. All data werecollected and plotted as the mean of triplicate points. In theseexperiments, the EC₅₀ was modeled in Prism as normalized activity, wherethe amount of AMC released in the absence of Salinosporamide Arepresents 100% activity. As before, the model chosen was a sigmoidaldose-response, with a variable slope.

Data revealed that Salinosporarnide A inhibited the PGPH activity inrabbit muscle 20S proteasomes with an EC₅o of 350 nM (FIG. 28). Areplicate experiment was performed, which gave a predicted EC₅₀ of 610nM. These results indicate that Salinosporamide A does block the invitro PGPH activity of purified rabbit muscle 20S proteasomes, albeitwith lower potency than seen towards the chymotrypsin-like activity.

Example 23 Inhibition of the Chymotrypsin-Like Activity of HumanErythrocyte 20S Proteasomes

The ability of Salinosporamide A (II-16) to inhibit thechymotrypsin-like activity of human erythrocyte 20S proteasomes wasassessed in vitro. The calculated EC₅₀ values ranged from 45 to 247 pM,and seemed to depend upon the lot of proteasomes tested (BIOMOL,Cat#SE-221). These data indicate that the inhibitory effect ofSalinosporamide A is not limited to rabbit skeletal muscle proteasomes.

Salinosporamide A was prepared as a 20 mM solution in DMSO and stored insmall aliquots at −80° C. The substrate, suc-LLVY-AMC, was prepared as a20 mM solution in DMSO and stored at −20° C. Human erythrocyte 20Sproteasomes were obtained from BIOMOL (Cat. # SE-221). The proteasomecan cleave suc-LLVY-AMC into suc-LLVY and free AMC and the activity canthen be determined by measuring the fluorescence of the released AMC(λ_(ex)=390 nm and λ_(em)=460 nm ). The proteasomes were activated bySDS and stored on ice as with the experiments using rabbit muscleproteasomes. Salinosporamide A was diluted in DMSO to generate a400-fold concentrated 8-point dilution series. The series was thendiluted 20-fold with assay buffer and pre-incubated with proteasomes at37° C. The reaction was initiated with substrate, and the release of AMCwas followed in a Fluoroskan microplate fluorimeter. Data were collectedand plotted as the mean of triplicate points. Data were capturedkinetically for 3 hours, and indicated that these reactions showedlinear kinetics in this time regime. The data were normalized toreactions performed in the absence of Salinosporamide A and modeled inPrism as a sigmoidal dose-response, variable slope.

Replicate experiments performed using human erythrocyte proteasomes fromseparate lots resulted in a range of EC₅₀ values between 45 and 250 pM(FIG. 29 shows a representative experiment). It has been reported that20S proteasomes purified from human erythrocytes are highlyheterogeneous in subunit composition. Claverol, et al., Mol CellProteomics 1:567 (2002). The variability in these experiments maytherefore be due to differences in the composition and activity of thehuman erythrocyte proteasome preparations. Regardless, these resultsindicate that the in vitro chymotrypsin-like activity of humanerythrocyte 20S proteasomes is sensitive to Salinosporamide A.

Example 24 Salinosporamide A (II- 16) Specificity

A possible mechanism by which Salinosporamide A inhibits the proteasomeis by the reaction of the β-lactone functionality of Salinosporamide Awith the active site threonine of the proteasome. This covalentmodification of the proteasome would block the active site, as thisresidue is essential for the catalytic activity of the proteasome.Fenteany, et al., J Biol Chem 273:8545 (1998). A structurally relatedcompound, Lactacystin, has been shown to also inhibit cathepsin A(Ostrowska, et al., Int J Biochem Cell Biol 32:747 (2000), Kozlowski, etal., Tumour Biol 22:211 (2001), Ostrowska, et al., Biochem Biophys ResCommun 234:729 (1997)) and TPPII (Geier, et al., Science 283:978 (1999))but not trypsin, chymotrypsin, papain, calpain (Fenteany, et al.,Science 268:726 (1995)), thrombin, or plasminogen activator (Omura, etal., J Antibiot (Tokyo) 44:113 (1991)). Similar studies were initiatedto explore the specificity of Salinosporamide A for the proteasome byevaluating its ability to inhibit the catalytic activity of aprototypical serine protease, chymotrypsin.

Salinosporamide A was prepared as a 20 mM solution in DMSO and stored insmall aliquots at −80° C. The substrate, suc-LLVY-AMC, was prepared as a20 mM solution in DMSO and stored at −20° C. Proteolytic cleavage ofthis substrate by either proteasomes or chymotrypsin liberates thefluorescent product AMC, which can be monitored in a fluorimeter(λ_(ex)=390 nm and λ_(em)=460 nm). Bovine pancreatic chymnotrypsin wasobtained from Sigma (Cat. #C-4129), and prepared as a 5 mg/ml solutionin assay buffer (10 mM HEPES, 0.5 mM EDTA, 0.05% Triton X-100, pH 7.5)daily. Immediately prior to the assay, the chymotrypsin was diluted to 1μg/ml (0.2 μg/well) in assay buffer and held on ice. Salinosporamide Awas diluted in DMSO to generate an 8-point dose-response curve. The highfinal Salinosporamide A concentrations needed to obtain completeinhibition of chymotrypsin required that the diluted enzyme be directlyadded to the compound dilution series. The inclusion of 1% DMSO (thefinal concentration of solvent in the test wells) into the reaction hadno significant effect on chymotrypsin activity towards this substrate.The reactions were pre-incubated for 5 minutes at 37° C. and thereactions were initiated by the addition of substrate. Data werecollected kinetically for one hour at 37° C. in the Fluoroskan andplotted as the mean of triplicate data points. The data were normalizedto reactions performed in the absence of Salinosporamide A, and modeledin Prism as a sigmoidal dose-response, variable slope. Normalized datafrom Salinosporamide A inhibition of the chymotrypsin-like activity ofrabbit 20S proteasomes has been included on the same graph.

The average inhibition observed in two experiments using SalinosporamideA pretreatment of chymotrypsin was 17.5 μM (FIG. 30 shows arepresentative experiment). The data indicate that there is a preferencefor Salinosporamide A-mediated inhibition of the in vitrochymotrypsin-like activity of proteasomes over inhibition of thecatalytic activity of chymotrypsin.

Thus, Salinosporamide A inhibits the chymotrypsin-like and PGPH activityof the proteasome. Preliminary studies indicate that Salinosporamide Aalso inhibits the trypsin-like activity of the proteasome with an EC₅₀value of ˜10 nM (data not shown).

Example 25 Inhibition of NF-κB-Mediated Luciferase Activity by FormulaeII-2, II-3, II-4, II-5A. II-5B, II-8C, II-13C, II-16, II-17, II-18,II-19, II-20, II-21, II-22, II-24C, II-25 and IV-3C; HEK293NF-κB/Luciferase Reporter Cell Line

The HEK293 NF-κB/luciferase reporter cell line is a derivative of thehuman embryonic kidney cell line (ATCC; CRL- 1573) and carries aluciferase reporter gene under the regulation of 5×NF-κB binding sites.The reporter cell line was routinely maintained in complete DMEM medium(DMEM plus 10%(v/v) Fetal bovine serum, 2 mM L-glutamine, 10 mM HEPESand Penicillin/Streptomycin at 100 IU/ml and 100 μg/ml, respectively)supplemented with 250 μg/ml G418. When performing the luciferase assay,the DMEM basal medium was replaced with phenol-red free DMEM basalmedium and the G418 was omitted. The cells were cultured in an incubatorat 37° C. in 5% CO₂ and 95% humidified air.

For NF-κB-mediated luciferase assays, HEK293 NF-κB/luciferase cells wereseeded at 1.5×10⁴ cells/well in 90 μl phenol-red free DMEM completemedium into Corning 3917 white opaque-bottom tissue culture plates. ForFormula II-2, Formula II-4, Formula II-5A, Formula II-16 and FormulaII-18, a 400 μM starting dilution was made in 100% DMSO and thisdilution was used to generate a 8-point half log dilution series. Thisdilution series was further diluted 40× in appropriate culture mediumand ten μl aliquots were added to the test wells in triplicate resultingin final test concentrations ranging from 1 μM to 320 pM. For FormulaII-3, Formula II-5B, Formula II-8C, Formula II-13C, Formula II-17,Formula II-20, Formula II-21, Formula II-22, Formula II-24C, FormulaII-25 and IV-3C, a 8 mM starting dilution was made in 100% DMSO and thesame procedure was followed as described above resulting in final testconcentrations ranging from 20 μM to 6.3 nM. For Formula II-19, a 127 μMstarting dilution was made in 100% DMSO and the final testconcentrations ranging from 317 nM to 0.1 nM. The plates were returnedto the incubator for 1 hour. After 1 hr pretreatment, 10 μl of a 50ng/ml TNF-α solution, prepared in the phenol-red free DMEM medium wasadded, and the plates were incubated for an additional 6 hr. The finalconcentration of DMSO was 0.25% in all samples.

At the end of the TNF-(X stimulation, 100 μl of Steady Lite HTSluciferase reagent (Packard Bioscience) was added to each well and theplates were left undisturbed for 10 min at room temperature beforemeasuring the luciferase activity. The relative luciferase units (RLU)were measured by using a Fusion microplate fluorometer (PackardBioscience). The EC₅₀ values (the drug concentration at which 50% of themaximal relative luciferase unit inhibition is established) werecalculated in Prism (GraphPad Software) using a sigmoidal dose response,variable slope model.

Inhibition of NF-κB Activation by Formulae II-2, II-3, II-4, II-5A,II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19, II-20, II-21, II-22,II-24C, II-25 and IV-3C

NF-κB regulates the expression of a large number of genes important ininflammation, apoptosis, tumorigenesis, and autoimmune diseases. In itsinactive form, NF-κB complexes with IκB in the cytosol and uponstimulation, IκB is phosphorylated, ubiquitinated and subsequentlydegraded by the proteasome. The degradation of IκB leads to theactivation of NF-κB and its translocation to the nucleus. The effects ofFormula II-2, Formula II-3, Formula II-4, Formula II-5A, Formula II-5B,Formula II-8C, Formula II-13C, Formula II-16, Formula II-17, FormulaII-18, Formula II-19, Formula II-20, Formula II-21, Formula II-22,Formula II-24C, Formula II-25 and Formula IV-3C on the activation ofNF-κB were evaluated by assessing the NF-κB-mediated luciferase activityin HEK293 NF-κB/Luc cells upon TNF-α stimulation.

Pretreatment of NF-κB/Luc 293 cells with Formula II-2, Formula II-4,Formula II-5A, Formula II-5B, Formula II-16, Formula II-17, FormulaII-18, Formula II-19, Formula II-20, Formula II-21, Formula II-22 andFormula II-24C resulted in a dose-dependent decrease of luciferaseactivity upon TNF-α stimulation. The mean EC₅₀ values to inhibitNF-κB-mediated luciferase activity are shown in Table 4 and demonstratethat compounds of Formula II-2, Formula II-4, Formula II-5A, FormulaIl-5B, Formula II-16, Formula II-17, Formula II-18, Formula II-19,Formula II-20, Formula II-21, Formula II-22 and Formula II-24C inhibitedNF-κB activity in this cell-based assay. TABLE 4 Mean EC₅₀ values ofFormulae II-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17,II-18, II-19, II-20, II-21, II-22, II-24C, II-25 and IV-3C fromNF-κB-mediated luciferase reporter gene assay Compound EC₅₀ (nM) FormulaII-2 82 Formula II-3 >20,000 Formula II-4 77.7 Formula II-5A 31.5Formula II-5B 270 Formula II-8C* >20,000 Formula II-13C >20,000 FormulaII-16 11.8 Formula II-17 876 Formula II-18 9.5 Formula II-19 8.5 FormulaII-20* 154 Formula II-21* 3,172 Formula II-22* 1,046 Formula II-24C*5,298 Formula II-25* >20,000 Formula IV-3C >20,000*n = 1

Example 26 Effect of Salinosporamide A on the NF-κB Signaling Pathway

Experiments were carried out to study the role of Salinosporamide A inthe NF-κB signaling pathway. A stable HEK293 clone (NF-κB/Luc 293) wasgenerated carrying a luciferase reporter gene under the regulation of5×NF-κB binding sites. Stimulation of this cell line with TNF-α leads toincreased luciferase activity as a result of NF-κB activation.

NF-κB/Luc 293 cells were pre-treated with 8-point half-log serialdilutions of Salinosporamide A (ranging from 1 μM to 317 pM) for 1 hourfollowed by a 6 hour stimulation with TNF-α (10 ng/mL). NF-κB inducibleluciferase activity was measured at 6 hours. Viability of NF-κB/Luc 293cells, after treatment with Salinosporamide A for 24 hr, was assessed bythe addition of resazurin dye, as previously described.

Pretreatment of NF-κB/Luc 293 cells with Salinosporamide A resulted in adose-dependent decrease of luciferase activity upon TNF-α stimulation(FIG. 31, right y-axis). The calculated EC₅₀ for inhibition ofNF-κB/luciferase activity was ˜7 nM. A cytotoxicity assay wassimultaneously performed, and showed that this concentration ofSalinosporamide A did not affect cell viability (FIG. 31, left y-axis).These representative data suggested that the observed decrease inluciferase activity by Salinosporamide A treatment was primarily due toan NF-κB mediated-signaling event rather than cell death.

Example 27

In addition to the NF-κB luciferase reporter gene assay, the effect ofSalinosporamide A on the levels of phosphorylated-IκBα and total IκBαwas evaluated by western blot. Endogenous protein levels were assessedin both HEK293 cells and the NF-κB/Luc 293 reporter clone.

Cells were pre-treated for 1 hour with Salinosporamide A at theindicated concentrations followed by stimulation with 10 ng/mL of TNF-αfor 30 minutes. Antibodies against total and phosphorylated forms ofIκBα were used to determine the endogenous level of each protein andanti-Tubulin antibody was used to confirm equal loading of protein.

As shown in FIG. 32, treatment of both cell lines with Salinosporamide Aat 50 and 500 nM not only reduced the degradation of total IκBα but alsoretained the phospho-IκBα level when stimulated with TNF-α. Theseresults strongly support the mechanism of action of Salinosporamide A asa proteasome inhibitor, which prevents the degradation of phosphorylatedIκBα upon TNF-α stimulation.

Example 28 Effect of Salinosporamide A on Cell Cycle Regulatory Proteins

The ubiquitin-proteasome pathway is an essential proteolytic systeminvolved in cell cycle control by regulating the degradation of cyclinsand cyclin-dependent kinase (Cdk) inhibitors such as p21 and p27.Pagano, et al., Science 269:682 (1995), Kisselev, et al., Chem Biol8:739 (2001), King, et al., Science 274:1652 (1996). Furthermore, p21and p27 protein levels are increased in the presence of proteasomeinhibitors. Fukuchi, et al., Biochim Biophys Acta 1451:206 (1999),Takeuchi, et al., Jpn J Cancer Res 93:774 (2002). Therefore, westernblot analysis was performed to evaluate the effect of Salinosporamide Atreatment on endogenous levels of p21 and p27 using the HEK293 cells andthe HEK293 NF-κB/Luciferase reporter clone.

The Western blots presented in FIG. 33 were reprobed using antibodiesagainst p21 and p27 to determine the endogenous level of each proteinand anti-Tubulin antibody was used to confirm equal loading of protein.

As shown in FIGS. 33A and 33B, preliminary results indicated that p21and p27 protein levels were elevated when both cell lines were treatedwith Salinosporamide A at various concentrations. Data showed thatSalinosporamide A acts by inhibiting proteasome activity therebypreventing the TNF-α induced activation of NF-κB. In addition, thisproteasomal inhibition results in the accumulation of the Cdkinhibitors, p21 and p27, which has been reported to sensitize cells toapoptosis. Pagano, et al., supra (1995), King, et al., supra (1996).

Example 29 Activation of Caspase-3 by Salinosporamide A (II-16)

To address whether Salinosporamide A induces apoptosis, its effect onthe induction of Caspase-3 activity was evaluated using Jurkat cells(American Type Culture Collection (ATCC) TIB-152, human acute T cellleukemia).

Jurkat cells were plated at 2×10⁶ cells/3 mL per well in a 6-well plateand incubated at 37° C., 5% (v/v) C0₂ and 95% (v/v) humidity.Salinosporamide A and Mitoxantrone (Sigma, St. Louis, Mo. Cat #M6545),were prepared in DMSO at stock concentrations of 20 mM and 40 mM,respectively. Mitoxantrone is a chemotherapeutic drug that inducesapoptosis in dividing and non-dividing cells via inhibition of DNAsynthesis and repair and was included as a positive control. Bhalla, etal., Blood 82:3133 (1993). Cells were treated with EC₅₀ concentrations(Table 5) and incubated 19 hours prior to assessing of Caspase-3activity. Cells treated with 0.25% DMSO served as the negative control.The cells were collected by centrifugation and the media removed. Cellpellets were processed for the Caspase-3 activity assay as described inthe manufacturer's protocol (EnzChek Caspase-3 Assay Kit from MolecularProbes (E-13183; see Appendix G, which form a part of this applicationand is also available at hypertext transfer protocol on the worldwideweb at “probes.com/media/pis/mp13183.pdf.”. In brief, cell pellets werelysed on ice, mixed with the EnzChek Caspase-3 components in a 96-wellplate, and then incubated in the dark for 30 minutes prior to readingfluorescence of cleaved benzyloxycarbonyl-DEVD-AMC using a PackardFusion with λ_(ex)=485 nm and λ_(em)=530 nm filters. Proteinconcentrations for lysates were determined using the BCA Protein AssayKit (Pierce) and these values were used for normalization.

Data from representative experiments indicate that Salinosporamide Atreatment of Jurkat cells results in cytotoxicity and activation ofCaspase-3 (Table 5, FIG. 34). TABLE 5 EC50 Values of Salinosporamide Aand Mitoxantrone Cytotoxicity against Jurkat Cells Jurkat Cells CompoundEC₅₀ (nM) % max cell kill Salinosporamide A 10 97 Mitoxantrone 50 99

Example 30 PARP Cleavage by Salinosporamide A in Jurkat Cells

In order to assess the ability of Salinosporamide A to induce apoptosisin Jurkat cells, cleavage of poly (ADP-ribose) polymerase (PARP) wasmonitored. PARP is a 116 kDa nuclear protein that is one of the mainintracellular targets of Caspase-3. Decker, et al., J Biol Chem 275:9043(2000), Nicholson, D. W, Nat Biotechnol 14:297 (1996). The cleavage ofPARP generates a stable 89 kDa product, and this process can bemonitored by western blotting. Cleavage of PARP by caspases is ahallmark of apoptosis, and as such serves as an excellent marker forthis process.

Jurkat cells were maintained in RPMI supplemented with 10% Fetal BovineSerum (FBS) at low density (2×10⁵ cells per mL) prior to the experiment.Cells were harvested by centrifugation, and resuspended in media to1×10⁶ cells per 3 mL. Twenty mL of the cell suspension were treated with100 nM Salinosporamide A (20 mM DMSO stock stored at −80° C.), and a 3mL aliquot removed and placed on ice for the T₀ sample. Three mLaliquots of the cell suspension plus Salinosporamide A were placed in6-well dishes and returned to the incubator. As a positive control forPARP cleavage, an identical cell suspension was treated with 350 nMStaurosporine, a known apoptosis inducer (Sigma S5921, 700 μM DMSO stockstored at −20° C.). Samples were removed at 2, 4, 6, 8, and 24 hrs inthe case of Salinosporamide A treated cells, and at 4 hrs for theStaurosporine control. For each time point, the cells were recovered bybrief centrifugation, the cells were washed with 400 μL of PBS, and thecells pelleted again. After removal of the PBS, the pellets were storedat −20° C. prior to SDS PAGE. Each cell pellet was resuspended in 100 μLof NuPAGE sample buffer (Invitrogen 46-5030) and 10 μL of each samplewere separated on 10% NuPAGE BIS-Tris gels (Invitrogen NB302). Afterelectrotransfer to nitrocellulose, the membrane was probed with a rabbitpolyclonal antibody to PARP (Cell Signaling 9542), followed by goatanti-rabbit alkaline phosphatase conjugated secondary antibody (Jackson11-055-045). Bound antibodies were detected colorimetrically usingBCIP/NBT (Roche 1681451).

The western blot presented in FIG. 35 shows the cleavage of PARP withinthe Jurkat cells in a time-dependent fashion. The cleaved form (denotedby the asterisk, *) appears in the treated cells between 2 and 4 hrsafter exposure to Salinosporamide A while the majority of the remainingPARP is cleaved by 24 hrs. The Staurosporine treated cells (St) showrapid cleavage of PARP with most of this protein being cleaved within 4hours. These data strongly suggest that Salinosporamide A can induceapoptosis in Jurkat cells.

Example 31 Anti-Anthrax Activity

In order to assay for the ability of Salinosporamide A or othercompounds to prevent cell death resulting from LeTx exposure, RAW264.7macrophage-like cells and recombinant LF and PA lethal toxin componentswere used as an in vitro model system assaying for cytotoxicity, asdescribed below.

RAW264.7 cells (ATCC #TIB-71) were adapted to and maintained in AdvancedDulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, Calif.)supplemented with 5% fetal bovine serum (ADMEM, Mediatech, Herndon, Va.)at 37° C. in a humidified 5% CO₂ incubator. Cells were plated overnightin ADMEM supplemented with 5% FBS at 37° C. in a humidified 5% CO₂incubator at a concentration of 50,000 cells/well in a 96-well plate.Alternatively, cells cultured in DMEM supplemented with 10% fetal calfserum were also used and found to be amenable to this assay. Media wasremoved the following morning and replaced with serum-free ADMEM with orwithout Salinosporamide A or Omuralide at doses ranging from 1 μM to 0.5nM for an 8-point dose-response. The compounds were prepared from a 1mg/mL DMSO stock solution and diluted to the final concentration inADMEM. After a 15 minute pre-incubation, 200 ng/mL LF or 400 ng/mL PAalone or in combination (LeTx) were added to cells. Recombinant LF andPA were obtained from List Biological Laboratories and stored as 1 mg/mLstock solutions in sterile water containing 1 mg/ml BSA at −80 ° C. asdescribed by the manufacturer. Cells were incubated for 6 hours at 37°C., followed by addition of Resazurin as previously described. Plateswere incubated an additional 6 hours prior to assessing cell viabilityby measuring fluorescence. The data are a summary of three experimentswith three to six replicates per experiment and are expressed as thepercent viability using the DMSO (negative) and the LeTx controls(positive) to normalize the data using the following equation: %viability=100*(observed OD-positive control)/(negative control-positivecontrol).

The data represented in FIG. 36 indicate that treatment withSalinosporamide A can prevent LeTx-induced cell death of macrophage-likeRAW264.7 cells in vitro. Treatment of RAW cells with either LF or PAalone or Salinosporamide A alone resulted in little reduction in cellviability, whereas treatment with LeTx resulted in approximately 0.27%cell viability as compared to controls. Salinosporamide A may enhancemacrophage survival by inhibiting the degradation of specific proteinsand decreasing the synthesis of cytokines, which will ultimately lead tothe inhibition of the lethal effects of anthrax toxins in vivo.

Although Salinosporamide A treatment alone produced very modestcytotoxicity at concentrations of 100 nM and above, treatment withlower, relatively non-toxic levels revealed a marked increase in RAW264.7 cell viability in LeTx treated cells (FIG. 36). For example, theSalinosporamide A+LeTx treated group showed 82% cell-viability whenpretreated with 12 nM Salinosporamide A, which was a concentration thatshowed 96% viability with Salinosporamide A alone. The average EC₅₀ forSalinosporamide A in these studies was 3.6 nM. In contrast, Omuralideshowed relatively little effect on cell viability until concentrationsof 1 μM were reached. Even at this high concentration of Omuralide, only37% viability was observed indicating that Salinosporamide A is a morepotent inhibitor of LeTx-induced RAW264.7 cell death. Consistent withthese data, Tang et. al., Infect Immun 67:3055 (1999), found that theEC₅₀ concentrations for MG132 and Lactacystin (the precursor toOmuralide) in the LeTx assay were 3 μM. Taken together, these datafurther illustrate that Salinosporamide A is a more potent inhibitor ofLeTx-induced cytotoxicity than any other compound described to date.

Salinosporamide A promoted survival of RAW264.7 cells in the presence ofLeTx indicating that this compound or it's derivatives may be a valuableclinical therapeutic for anthrax. In addition, it is worth noting thatSalinosporamide A is much less cytotoxic on RAW 264.7 cells than formany tumor cells.

Example 32 Activity of Salinosporamide A Against Multiple Myeloma andProstate Cancer Cell Lines

NF-?B appears to be critical to the growth and resistance to apoptosisin Multiple Myeloma and has also been reported to be constitutivelyactive in various prostate cancer cell lines (Hideshima T et al. 2002,Shimada K et al. 2002 and Palayoor S T et al. 1999). NF-κB activity isregulated by the proteasomal degradation of its inhibitor IκBα. SinceSalinosporamide A has been shown to inhibit the proteasome in vitro andto interfere with the NF-κB signaling pathway, the activity ofSalinosporamide A against the multiple myeloma cell line RPMI 8226 andthe prostate cancer cell lines PC-3 and DU 145 was evaluated.

EC₅₀ values were determined in standard growth inhibition assays usingResazurin dye and 48 hour of drug exposure. Results from 2-5 independentexperiments (Table 6) show that the EC₅₀ values for Salinosporamide Aagainst RPMI 8226 and the prostate cell lines range from 10-37 nM. TABLE6 EC₅₀ values of Salinosporamide A (II-16) against Multiple Myeloma andProstate Tumor cell lines RPMI 8226 (n = 5) DU 145 (n = 3) EC₅₀ % EC₅₀ %PC-3 (n = 2) (nM), cytotoxicity, (nM), cytotoxicity, EC₅₀ % Compoundmean ± SD mean ± SD mean ± SD mean ± SD (nM) cytotoxicitySalinosporamide A 10 ± 3 94 ± 1 37 ± 10 75 ± 4 31, 25 88, 89

The ability of Salinosporamide A to induce apoptosis in RPMI 8226 andPC-3 cells was evaluated by monitoring the cleavage of PARP andPro-Caspase 3 using western blot analysis. Briefly, PC-3 and RPMI 8226cells were treated with 100 nM Salinosporamide A (2345R01) for 0, 8 or24 hours. Total protein lysates were made and 20 μg of the lysates werethen resolved under reducing/denaturing conditions and blotted ontonitrocellulose. The blots were then probed with anti-PARP oranti-caspase 3 antibodies followed by stripping and reprobing with ananti-actin antibody.

Results of these experiments illustrate that Salinosporamide A treatmentof RPMI 8226 cells leads to the cleavage of PARP and Pro-caspase 3 in atime-dependent manner (FIG. 37). RPMI 8226 cells seem to be moresensitive to Salinosporamide A than PC-3 cells since the induction ofPARP cleavage is already noticeable at 8 hours and complete by 24 hours.In contrast, in PC-3 cells the cleavage of PARP is noticeable at 24hours, while the cleavage of Pro-Caspase 3 is not detected in thisexperiment (FIG. 37).

RPMI 8226 cells were used to evaluate the effect of treating the cellsfor 8 hours with various concentrations of Salinosporamide A. Briefly,RPMI 8226 cells were treated with varying concentrations ofSalinosporamide A (2345R01) for 8 hours and protein lysates were made.25 μg of the lysates were then resolved under reducing/denaturingconditions and blotted onto nitrocellulose. The blots were then probedwith anti-PARP or anti-caspase 3 antibodies followed by stripping andreprobing with an anti-actin antibody. FIG. 38 demonstrates thatSalinosporamide A induces a dose-dependent cleavage of both PARP andPro-Caspase 3.

Example 33 Growth Inhibition of Human Multiple Myeloma by Formulae II-2,II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,II-20, and IV-3C; RPMI 8226 and U266 Cells

The human multiple myeloma cell lines, RPMI 8226 (ATCC; CCL-155) andU266 (ATCC; TIB-196) were maintained in appropriate culture media. Thecells were cultured in an incubator at 37° C. in 5% CO₂ and 95%humidified air.

For cell growth inhibition assays, RPMI 8226 cells and U266 were seededat 2×10⁴ and 2.5×10⁴ cells/well respectively in 90 μl complete mediainto Corning 3904 black-walled, clear-bottom tissue culture plates. 20mM stock solutions of the compounds were prepared in 100% DMSO,aliquoted and stored at −80° C. The compounds were serially diluted andadded in triplicate to the test wells. The final concentration range ofFormula II-3, II-8C, II-5B, II-13C, II-17, IV-3C and II-20 were from 20μM to 6.32 nM. The final concentration of Formula II-16, II-18 and II-19ranged from 632 nM to 200 pM. The final concentration range of FormulaII-2, II-4 and II-5A were from 2 μM to 632 pM. The final concentrationof DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigmna-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (generated byXLfit 3.0, ID Business Solutions Ltd). The data are summarized in Tables13 and 15.

Example 34 Salinosporamide A (II-16) Retains Activity Against theMulti-Drug Resistant Cell Lines MES-SA/Dx5 and HL-60/MX2

The EC₅₀ values of Salinosporamide A against the human uterine sarcomaMES-SA cell line and its multidrug-resistant derivative MES-SA/Dx5 weredetermined to evaluate whether Salinosporamide A retains activityagainst a cell line overexpressing the P-glycoprotein efflux pump.Paclitaxel, a known substrate for the P-glycoprotein pump was includedas a control. TABLE 7 EC₅₀ values of Salinosporamide A against MES-SAand the drug-resistant derivative MES-SA/Dx5 MES-SA MES-SA/Dx5 EC₅₀(nM), % cytotoxicity, EC₅₀ (nM), % cytotoxicity, Fold mean mean ± SDmean ± SD mean ± SD change Salinosporamide A 20 ± 5 94 ± 1 23 ± 1 92 ± 21.2 Paclitaxel  5 ± 2 63 ± 7 2040 ± 150 78 ± 1 408

Results from these growth inhibition assays (Table 7) show that, asexpected, Paclitaxel did not retain its activity against MES-SA/Dx5cells as reflected by the 408 fold increase in the EC₅₀ values. EC₅₀values for Salinosporamide A against MES-SA and MES-SA/Dx5 were similar.This illustrates that Salinosporamide A is able to inhibit the growth ofthe multi-drug resistant cell line MES-SA/Dx5 suggesting thatSalinosporamide A does not seem to be a substrate for the P-glycoproteinefflux pump.

In addition, Salinosporamide A was evaluated against HL-60/MX2, the drugresistant derivative of the human leukemia cell line, HL-60,characterized by having a reduced Topoisomerase II activity andconsidered to have atypical multidrug resistance. EC₅₀ values for growthinhibition were determined for Salinosporamide A against the HL-60 andHL-60/MX2. The DNA binding agent Mitoxantrone was included as a control,as HL-60/MX2 cells are reported to be resistant to this chemotherapeuticagent (Harker W. G. et al. 1989). TABLE 8 EC₅₀ values of SalinosporamideA against HL-60 and the drug resistant derivative HL-60/MX2 HL-60HL-60/MX2 Fold EC₅₀ (nM) % cytotoxicity EC₅₀ (nM) % cytotoxicity changeSalinosporamide A 27, 30 88, 91   28, 25 84, 89 1.0, 0.8 Mitoxantrone59, 25 98, 100 1410, 827 98, 99 24, 33

The data in Table 8 reveals that Salinosporamide A was able to retainits activity against HL-60/MX2 cells relative to HL-60 cells, indicatingthat Salinosporamide A is active in cells expressing reducedTopoisomerase II activity. In contrast, Mitoxantrone was about 29 foldless active against HL-60/MX2 cells.

Example 35 Salinosporamide A and Several Analogs: Structure ActivityRelationship

To establish an initial structure activity relationship (SAR) forSalinosporamide A, a series of Salinosporamide A analogs were evaluatedagainst the multiple myeloma cell line RPMI 8226. EC₅₀ values weredetermined in standard growth inhibition assays using Resazurin dye and48 hour of drug exposure.

The results of this initial series of SAR (Table 9) indicate that theaddition of a halogen group to the ethyl group seems to enhance thecytotoxic activity. TABLE 9 Initial SAR series of Salinosporamide AEC₅₀, Compound μM (mean ± % Cytotoxicity No. Molecular Structure SD)(mean ± SD) II-16

0.007 ± 0.0001 94 ± 0 II-17

2.6, 2.3 94, 95 II-18

0.017, 0.022 94, 94Where n>2, mean ± standard deviation was determined

Example 36 In vivo Biology Maximum Tolerated Dose (MTD) Determination

In vivo studies were designed to determine the MTD of Salinosporamide Awhen administered intravenously to female BALB/c mice.

BALB/c mice were weighed and various Salinosporamide A concentrations(ranging from 0.01 mg/kg to 0.5 mg/kg) were administered intravenouslyas a single dose (qdx1) or daily for five consecutive days (qdx5).Animals were observed daily for clinical signs and were weighedindividually twice weekly until the end of the experiment (maximum of 14days after the last day of dosing). Results are shown in Table 11 andindicate that a single intravenous Salinosporamide A dose of up to 0.25mg/kg was tolerated. When administered daily for five consecutive days,concentrations of Salinosporamide A up to 0.1 mg/kg were well tolerated.No behavioral changes were noted during the course of the experiment.TABLE 11 MTD Determination of Salinosporamide A in female BALB/c MiceGroup Dose (mg/kg) Route/Schedule Deaths/Total Days of Death 1 0.5 i.v.;qdx1 3/3 3, 3, 4 2 0.25 i.v.; qdx1 0/3 3 0.1 i.v.; qdx1 0/3 4 0.05 i.v.;qdx1 0/3 5 0.01 i.v.; qdx1 0/3 6 0 i.v.; qdx1 0/3 7 0.5 i.v.; qdx5 3/34, 6, 7 8 0.25 i.v.; qdx5 3/3 4, 5, 5 9 0.1 i.v.; qdx5 0/3 10 0.05 i.v.;qdx5 0/3 11 0.01 i.v.; qdx5 0/3 12 0 i.v.; qdx5 0/3

Example 37 Preliminary Assessment of Salinosporamide A Absorption,Distribution, Metabolism and Elimination (ADME) Characteristics

Studies to initiate the evaluation of the ADME properties ofSalinosporamide A were performed. These studies consisted of solubilityassessment, LogD^(7.4) determination and a preliminary screen to detectcytochrome P450 enzyme inhibition. Results from these studies showed anestimated solubility of Salinosporamide A in PBS (pH 7.4) of 9.6 μM (3μg/ml) and a LogD^(7.4) value of 2.4. This LogD^(7.4) value is withinthe accepted limits compatible with drug development (LogD^(7.4)<5.0)and suggests oral availability. Results from the preliminary P450inhibition screen showed that Salinosporamide A, when tested at 10 μM,showed no or low inhibition of all P450 isoforms: CYP1A2, CYP2C9 andCYP3A4 were inhibited by 3%, 6% and 6% respectively, while CYP2D6 andCYP2C19 were inhibited by 19% and 22% respectively.

Example 38 Salinosporamide A and its Effects in vivo on Whole BloodProteasome Activity

Salinosporamide A was previously demonstrated to be a potent andspecific inhibitor of the proteasome in vitro, with an IC₅₀ of 2 nMtowards the chymotrypsin-like activity of purified 20S proteasomes. Tomonitor the activity of Salinosporamide A in vivo, a rapid andreproducible assay (adapted from Lightcap et al. 2000) was developed toassess the proteosome activity in whole blood.

In brief, frozen whole blood samples were thawed on ice for one hour,and resuspended in 700 μL of ice cold 5 mM EDTA, pH 8.0 in order to lysethe cells by hypotonic shock. This represents approximately 2-3 timesthe volume of the packed whole blood cells. Lysis was allowed to proceedfor one hour, and the cellular debris was removed by centrifuigation at14,000×g for 10 minutes. The supernatant (Packed Whole Blood Lysate,PWBL) was transferred to a fresh tube, and the pellet discarded. Proteinconcentration of the PWBL was determined by BCA assay (Pierce) using BSAas a standard. Approximately 80% of the samples had a total proteinconcentration between 800 and 1200 μg/mL.

Proteasome activity was determined by measuring the hydrolysis of afluorogenic substrate specific for the chymotrypsin-like activity ofproteasomes (suc-LLVY-AMC, Bachem Cat. 1-1395). Control experimentsindicated that >98% of the hydrolysis of this peptide in these extractsis mediated by the proteasome. Assays were set up by mixing 5 μL of aPWBL from an animal with 185 μL of assay buffer (20 mM HEPES, 0.5 mMEDTA, 0.05% Triton X-100, 0.05% SDS, pH 7.3) in Costar 3904 plates.Titration experiments revealed there is a linear relationship betweenprotein concentration and hydrolysis rate if the protein concentrationin the assay is between 200 and 1000 μg. The reactions were initiated bythe addition of 10 μL of 0.4 mM suc-LLVY-AMC (prepared by diluting a 10mM solution of the peptide in DMSO 1:25 with assay buffer), andincubated in a fluorometer (Labsystems Fluoroskan) at 37° C. Hydrolysisof the substrate results in the release of free AMC, which was measuredfluorometrically by using λ_(ex)=390 nm and λ_(em)=460 nm. The rate ofhydrolysis in this system is linear for at least one hour. Thehydrolysis rate of each sample is then normalized to relativefluorescent units per milligram of protein (RFU/mg).

To explore the in vivo activity of Salinosporamide A, male Swiss-Webstermice (5 per group, 20-25 g in weight) were treated with variousconcentrations of Salinosporamide A. Salinosporamide A was administeredintravenously and given its LogD^(7.4) value of 2.4, suggestive of oralavailability, Salinosporamide A was also administered orally.Salinosporamide A dosing solutions were generated immediately prior toadministration by dilution of Salinosporamide A stock solutions (100%DMSO) using 10% solutol yielding a final concentration of 2% DMSO. Thevehicle control consisted of 2% DMSO in 10% solutol. One group ofanimals was not dosed with either vehicle or Salinosporamide A in orderto establish a baseline for proteasome activity. Salinosporamide A orvehicle was administered at 10 mL/kg and ninety minutes afteradministration the animals were anesthetized and blood withdrawn bycardiac puncture. Packed whole blood cells were collected bycentrifugation, washed with PBS, and re-centrifuged. All samples werestored at −80° C. prior to the evaluation of the proteasome activity.

In order to be certain that the hydrolysis of the substrate observed inthese experiments was due solely to the activity of the proteasome, doseresponse experiments on the extracts were performed using the highlyspecific proteasomal inhibitor Epoxomicin. PWBL lysates were diluted1:40 in assay buffer, and 180 μL were added to Costar 3904 plates.Epoxomicin (Calbochem Cat. 324800) was serially diluted in DMSO togenerate an eight point dose response curve, diluted 1:50 in assaybuffer, and 10 μL added to the diluted PWBL in triplicate. The sampleswere preincubated for 5 minutes at 37° C., and the reactions initiatedwith substrate as above. The dose response curves were analyzed inPrism, using a sigmoidal dose response with variable slope as a model.

FIG. 40 is a scatter plot displaying the normalized proteasome activityin PWBL's derived from the individual mice (5 mice per group). In eachgroup, the horizontal bar represents the mean normalized activity. Thesedata show that Salinosporamide A causes a profound decrease inproteasomal activity in PWBL, and that this inhibition is dosedependent. In addition, these data indicate that Salinosporamide A isactive upon oral administration.

The specificity of the assay was shown by examining the effect of aknown proteasome inhibitor, Epoxomicin, on hydrolysis of the peptidesubstrate. Epoxomicin is a peptide epoxide that has been shown to highlyspecific for the proteasome, with no inhibitory activity towards anyother known protease (Meng et al., 1999). Lysates from a vehicle controland also from animals treated intravenous (i.v.) with 0.1 mg/kgSalinosporamide A were incubated with varying concentration ofEpoxomicin, and IC₅₀ values were determined. Palayoor et al., Oncogene18:7389-94 (1999). As shown in FIG. 41, Epoxomicin caused a dosedependent inhibition in the hydrolysis of the proteasome substrate. TheIC₅₀ obtained in these experiments matches well with the 10 nM valueobserved using purified 20S proteasomes in vitro (not shown). These dataalso indicate that the remaining activity towards this substrate inthese lysates prepared from animals treated with 0.1 mg/kgSalinosporamide A is due to the proteasome, and not some other protease.The residual activity seen in extracts treated with high doses ofEpoxomicin is less than 2% of the total signal, indicating that over 98%of the activity observed with suc-LLVY-AMC as a substrate is due solelyto the activity of the proteasomes present in the PWBL.

Comparison of intra-run variation in baseline activity and the abilityof Salinosporamide A to inhibit proteasomal activity was also assessed.In FIG. 42, the results of separate assays run several weeks apart areshown. Qureshi, et al., J. Immunol. 171(3): 1515-25 (2003). For clarity,only the vehicle control and matching dose results are shown. Whilethere was some variation in the proteasomal activity in PWBL derivedfrom individual animals in the control groups, the overall mean was verysimilar between the two groups. The animals treated with SalinosporamideA (0.1 mg/kg i.v.) also show very similar residual activity and averageinhibition. This suggests that results between assays can be comparedwith confidence.

Example 39 Inhibition of in vivo LPS-Induced TNF by Salinosporamide A

Studies suggest that the proteasome plays a role in the activation ofmany signaling molecules, including the transcription factor NF-κB viaprotealytic degradation of the inhibitor of NF-κB (IκB). LPS signalingthrough the TLR4 receptor activates NF-κB and other transcriptionalregulators resulting in the expression of a host of proinflammatorygenes like TNF, IL-6, and IL-1β. The continued expression ofproinflammatory cytokines has been identified as a major factor in manydiseases. Inhibitors of TNF and IL-1β have shown efficacy in manyinflammation models including the LPS murine model, as well as animalmodels of rheumatoid arthritis and inflammatory bowel disease. Recentstudies have suggested that inhibition of the proteasome can preventLPS-induced TNF secretion (Qureshi et al., 2003). These data suggestthat Salinosporamide A, a novel potent proteasome inhibitor, may preventTNF secretion in vivo in the high-dose LPS murine model.

To assess the ability of Salinosporamide A to inhibit in vivoLPS-induced plasma TNF levels in mice, in vivo studies were initiated atBolderBioPATH, Inc. in Boulder, Colo. The following methods outline theprotocol design for these studies.

Male Swiss Webster mice (12/group weighing 20-25 g) were injected withLPS (2 mg/kg) by the i.p. route. Thirty minutes later, mice wereinjected i.v. (tail vein) with Salinosporamide A at 2.5 mg/kg afterapproximately 5 minutes under a heat lamp. Ninety minutes after LPSinjection, the mice were anesthetized with Isoflurane and bled bycardiac puncture to obtain plasma. Remaining blood pellet was thenresuspended in 500 μL of PBS to wash away residual serum proteins andcentrifuged again. Supernatant was removed and blood pellet frozen foranalysis of proteasome inhibition in packed whole blood lysate. TABLE 12Time Group ID Group n = 0 min +30 min No injections/baseline 1 5Saline + solutol vehicle 2 5 saline Saline + solutol vehicles 3 5 salineSolutol/DMSO LPS i.p./Vehicle (−30 min) 4 12 LPS LPS i.p./Vehicle (+30m) 5 12 LPS Solutol/DMSO saline/Salinosporamide A 6 12 saline (−30 min)0.25 mg/kg saline/Salinosporamide A 7 12 saline 0.25 mg/kg (+30 m) 0.25mg/kg LPS/Salinosporamide A (−30 min) 8 12 LPS 0.25 mg/kgLPS/Salinosporamide A (+30 m) 9 12 LPS 0.25 mg/kg 0.25 mg/kg

Dosing solutions were prepared using a 10 mg/mL Salinosporamide A stocksolution in 100% DMSO. A 10% solutol solution was prepared by dilutingw/w with endotoxin-free water and a 1:160 dilution was made of the 10mg/ml Salinosporamide A stock. Animals were dosed i.v. with 4 ml/kg. Avehicle control solution was also prepared by making the same 1:160dilution with 100% DMSO into 10% solutol solution giving a finalconcentration of 9.375% solutol in water and 0.625% DMSO. Measurementsof plasma TNF were performed using the Biosource mTNF Cytoset kit(Biosource Intl., Camarillo, Calif.; catalog #CMC3014) according tomanufacturer's instructions. Samples were diluted 1:60 for the assay.

Data from two independent experiments with at least ten replicateanimals per group indicated that treatment with 0.125 or 0.25 mg/kgSalinosporamide A decreased LPS-induced TNF secretion in vivo. Arepresentative experiment is shown in FIG. 43. These data reveal thattreatment of animals with 0.25 mg/kg Salinosporamide A thirty minutesafter 2 mg/kg LPS injection resulted in significant reduction in serumTNF levels. Packed whole blood samples were also analyzed for ex vivoproteasome inhibition revealing 70±3% inhibition in animals treated with0.125 mg/kg and 94±3% in animals treated with 0.25 mg/kg. No significantdifferences were seen in proteasome inhibition in animals treated withor without LPS. Salinosporamide A reduces LPS-induced plasma TNF levelsby approximately 65% when administered at 0.125 or 0.25 mg/kg i.v. intomice 30 minutes post-LPS treatment.

Example 40 Potential in vitro Chemosensitizing Effects ofSalinosporamide A

Chemotherapy agents such as CPT-11 (Irinotecan) can activate thetranscription factor nuclear factor-kappa B (NF-?B) in human coloncancer cell lines including LoVo cells, resulting in a decreased abilityof these cells to undergo apoptosis. Cusack, et al., Cancer Res 61:3535(2001). In unstimulated cells, NF-?B resides in the cytoplasm in aninactive complex with the inhibitory protein IκB (inhibitor of NF-κB).Various stimuli can cause IκB phosphorylation by IκB kinase, followed byubiquitination and degradation of IκB by the proteasome. Following thedegradation of IκB, NF-κB translocates to the nucleus and regulates geneexpression, affecting many cellular processes, including upregulation ofsurvival genes thereby inhibiting apoptosis.

The recently approved proteasome inhibitor, Velcade™ (PS-341; MillenniumPharmaceuticals, Inc.), is directly toxic to cancer cells and can alsoenhance the cytotoxic activity of CPT-11 against LoVo cells in vitro andin a LoVo xenograft model by inhibiting proteasome induced degradationof I?B. Adams, J., Eur J Haematol 70:265 (2003). In addition, Velcade™was found to inhibit the expression of proangiogenicchemokines/cytokines GRO-α and VEGF in squamous cell carcinoma,presumably through inhibition of the NF-κB pathway. Sunwoo, et al., ClinCancer Res 7:1419 (2001). The data indicate that proteasome inhibitionmay not only decrease tumor cell survival and growth, but alsoangiogenesis.

Example 41 Growth Inhibition of Colon, Prostate, Breast, Lung, Ovarian,Multiple Myeloma and Melanoma

Human colon adenocarcinoma (HT-29; HTB-38), prostate adenocarcinoma(PC-3; CRL-1435), breast adenocarcinoma (MDA-MB-231; HTB-26), non-smallcell lung carcinoma (NCI-H292; CRL-1848), ovarian adenocarcinoma(OVCAR-3; HTB-161), multiple myeloma (RPMI 8226; CCL-155), multiplemyeloma (U266; TIB-196) and mouse melanoma (B16-F10; CRL-6475) cellswere all purchased from ATCC and maintained in appropriate culturemedia. The cells were cultured in an incubator at 37° C. in 5% CO₂ and95% humidified air.

For cell growth inhibition assays, HT-29, PC-3, MDA-MB-231, NCI-H292,OVCAR-3 and B16-F10 cells were seeded at 5×10³, 5×10³, 1×10⁴, 4×10³,1×10⁴ and 1.25×10³ cells/ well respectively in 90 μl complete media into96 well (Corning; 3904) black-walled, clear-bottom tissue culture platesand the plates were incubated overnight to allow cells to establish andenter log phase growth. RPMI 8226 and U266 cells were seeded at 2×10⁴and 2.5×10⁴ cells/well respectively in 90 μl complete media into 96 wellplates on the day of the assay. 20 mM stock solutions of the compoundswere prepared in 100% DMSO and stored at −80° C. The compounds wereserially diluted and added in triplicate to the test wells.Concentrations ranging from 6.32 μM to 632 μM were tested for II-2 andII-4. II-3 and II-17 were tested at concentrations ranging from 20 μM to6.32 nM. Formula II-18 and II-19 were tested at concentrations rangingfrom 2 μM to 200 μM. Formula II-5A and Formula II-5B were tested atfinal concentrations ranging from 2 μM to 632 μM and 20 μM to 6.32 nMrespectively. The plates were returned to the incubator for 48 hours.The final concentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (XLfit 3.0,ID Business Solutions Ltd). Where the maximum inhibition of cell growthwas less than 50%, an EC₅₀ value was not determined.

The data in Table 13 summarize the growth inhibitory effects of FormulaeII-2, II-3, II-5A, II-5B, II-17, II-18 and II-19 against the humancolorectal carcinoma, HT-29, human prostate carcinoma, PC-3, humanbreast adenocarcinoma, MDA-MB-231, human non-small cell lung carcinoma,NCI-H292, human ovarian carcinoma, OVCAR-3, human multiple myelomas,RPMI 8226 and U266 and murine melanoma B16-F10 cell lines. TABLE 13 EC₅₀values of Formulae II-2, II-3, II-4, II-5A, II-5B, II-17, II-18 andII-19 against various tumor cell lines EC₅₀ (nM)* Cell line II-2 II-3II-4 II-5A II-5B II-17 II-18 II-19 HT-29 129 ± 21  >20000 132 ± 36 851070 >20000 18 ± 7.8 13 PC-3 284 ± 110 >20000 204 ± 49 97 1330 >20000 35± 5.6 27 MDA-MB-231 121 ± 23  >20000 114 ± 4  66 1040 5900 ± 601 16 ±2.8 17 NCI-H292 322 >20000 192 90 >20000 >20000 29 31 395 >20000213 >20000 41 OVCAR-3 188 >20000 >6320 NT NT >20000 >2000 NT251 >6320 >20000 >2000 RPMI 8226 49 >20000 57 36 326 6200 6.3 5.945 >20000 51 29 328 3500 6.3 7.1 U266 39 >20000 39 10 118 1620 4.2 3.232 >20000 34  9 111 1710 4.2 3.4 B16-F10 194 >20000 163 NT NT 10500 19NT 180 >20000 175 10300 36*Where n = 3, mean ± standard deviation is presented; NT = not tested

The EC₅₀ values indicate that the Formulae II-2, II-4 and II-18 werecytotoxic against the HT-29, PC-3, MDA-MB-231, NCI-H292, RPMI 8226, U266and B16-F10 tumor cell lines. II-2 was also cytotoxic against theOVCAR-3 tumor cells. Formula II-17 was cytotoxic against MDA-MB-23 1,RPMI 8226, U266 and B16-F10 tumor cell lines. Formulae II-5A, II-5B andII-19 were cytotoxic against HT-29, PC-3, MDA-MB-231, RPMI 8226 and U266tumor cells. Formula II-5A and II-19 were also cytotoxic againstNCI-H292 tumor cells.

The data in Table 15 summarize the growth inhibitory effects of FormulaeII-2, II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18,II-19, IV-3C and Formula II-20 against the human multiple myeloma celllines, RPMI 8226 and U266. TABLE 15 Mean EC₅₀ values of Formulae II-2,II-3, II-4, II-5A, II-5B, II-8C, II-13C, II-16, II-17, II-18, II-19,IV-3C and Formula II-20 against RPMI 8226 and U266 cells RPMI 8226 U266Compound EC₅₀ (nM) EC₅₀ (nM) Formula II-17 4800 1670 Formula II-16 7.04.1 Formula II-18 6.3 4.2 Formula II-2 47 36 Formula II-3 >20000 >20000Formula II-4 54 36 Formula II-5A 33 10 Formula II-5B 327 115 FormulaII-8C >20000 >20000 Formula II-13C >20000 >20000 Formula II-19 6.5 3.3Formula IV-3C >20000 8020 Formula II-20* 10500 3810*n = 1

The EC₅₀ values indicate that Formulae II-2, II-4, II-5A, II-5B, II-16,II-17, II-18, II-19 and II-20 were cytotoxic against RPMI 8226 and U266cells. Formula IV-3C was cytotoxic against U266 cells

Example 42 Growth Inhibition of MES-SA, MES-SA/Dx5, HL-60 and HL-60/MX2Tumor Cell Lines

Human uterine sarcoma (MES-SA; CRL-1976), its multidrug resistantderivative (MES-SA/Dx5; CRL-1977), human acute promyelocytic leukemiacells (HL-60; CCL-240) and its multidrug resistant derivative(HL-60/MX2; CRL-2257) were purchased from ATCC and maintained inappropriate culture media. The cells were cultured in an incubator at37° C. in 5% CO₂ and 95% humidified air.

For cell growth inhibition assays, MES-SA and MES-SA/Dx5 cells were bothseeded at 3×10³ cells/ well in 90 μl complete media into 96 well(Corning; 3904) black-walled, clear-bottom tissue culture plates and theplates were incubated overnight to allow cells to establish and enterlog phase growth. HL-60 and HL-60/MX2 cells were both seeded at 5×10⁴cells/ well in 90 μl complete media into 96 well plates on the day ofcompound addition. 20 mM stock solutions of the compounds were preparedin 100% DMSO and stored at −80° C. The compounds were serially dilutedand added in triplicate to the test wells. Concentrations ranging from6.32 μM to 2 nM were tested for II-2 and II-4. II-3 and II-17 weretested at concentrations ranging from 20 μM to 6.32 nM. Compound II-18was tested at concentrations ranging from 2 μM to 632 pM. The plateswere returned to the incubator for 48 hours. The final concentration ofDMSO was 0.25% in all samples.

Following 48 hours of drug exposure, 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free phosphatebuffered saline was added to each well and the plates were returned tothe incubator for 3-6 hours. Since living cells metabolize Resazurin,the fluorescence of the reduction product of Resazurin was measuredusing a Fusion microplate fluorometer (Packard Bioscience) withλ_(ex)=535 nm and λ_(em)=590 nm filters. Resazurin dye in medium withoutcells was used to determine the background, which was subtracted fromthe data for all experimental wells. The data were normalized to theaverage fluorescence of the cells treated with media +0.25% DMSO (100%cell growth) and EC₅₀ values (the drug concentration at which 50% of themaximal observed growth inhibition is established) were determined usinga standard sigmoidal dose response curve fitting algorithm (XLfit 3.0,ID Business Solutions Ltd). Where the maximum inhibition of cell growthwas less than 50%, an EC₅₀ value was not determined.

The multidrug resistant MES-SA/Dx5 tumor cell line was derived from thehuman uterine sarcoma MES-SA tumor cell line and expresses elevatedP-Glycoprotein (P-gp), an ATP dependent efflux pump. The data in Table16 summarize the growth inhibitory effects of Formulae II-2, II-3, II-4,II-17 and II-18 against MES-SA and its multidrug resistant derivativeMES-SA/Dx5. Paclitaxel, a known substrate of the P-gp pump was includedas a control. TABLE 16 EC₅₀ values of Formulae II-2, II-3, II-4, II-17and II-18 against MES-SA and MES-SA/Dx5 tumor cell lines EC₅₀ (nM) FoldCompound MES-SA MES-SA/Dx5 change* II-2 193 220 1.0 155 138II-3 >20000 >20000 NA >20000 >20000 II-4 163 178 0.9 140 93 II-17 92309450 0.8 12900 7530 II-18 22 32 1.2 17 14 Paclitaxel 5.6 2930 798 4.65210*Fold change = the ratio of EC₅₀ values (MES-SA/Dx5:MES-SA)

The EC₅₀ values indicate that II-2, II-4, II-17 and II-18 have cytotoxicactivity against both MES-SA and MES-SA/Dx5 tumor cell lines. Themultidrug resistant phenotype was confirmed by the observation thatPaclitaxel was ˜800 times less active against the resistant MES-SA/Dx5cells.

HL-60/MX2 is a multidrug resistant tumor cell line derived from thehuman promyelocytic leukemia cell line, HL-60 and expresses reducedtopoisomerase II activity. The data presented in Table 17 summarize thegrowth inhibitory effects of Formulae II-2, II-3, II-4, II-17 and II-18against HL-60 and its multidrug resistant derivative HL-60/MX2.Mitoxantrone, the topoisomerase II targeting agent was included as acontrol. TABLE 17 EC₅₀ values of Formulae II-2, II-3, II-4, II-17 andII-18 against HL-60 and HL-60/MX2 tumor cell lines EC₅₀ (nM) FoldCompound HL-60 HL-60/MX2 change* II-2 237 142 0.7 176 133II-3 >20000 >20000 NA >20000 >20000 II-4 143 103 0.8 111 97II-17 >20000 >20000 NA II-18 27 19 0.7 23 18 Mitoxantrone 42 1340 30.640 1170*Fold change = the ratio of EC₅₀ values (HL-60/MX2:HL-60)

The EC₅₀ values indicate that II-2, II-4 and II-18 retained cytotoxicactivity against both HL-60 and HL-60/MX2 tumor cell lines. Themultidrug resistant phenotype was confirmed by the observation thatMitoxantrone was ˜30 times less active against the resistant HL-60/MX2cells.

Example 43 Inhibition of NF-κB-Mediated Luciferase Activity: HEK293NF-κB/Luciferase Reporter Cell Line

The HEK293 NF-κB/luciferase reporter cell line is a derivative of thehuman embryonic kidney cell line (ATCC; CRL-1573) and carries aluciferase reporter gene under the regulation of 5×NF-κB binding sites.The reporter cell line was routinely maintained in complete DMEM medium(DMEM plus 10%(v/v) Fetal bovine serum, 2 mM L-glutamine, 10 mM HEPESand Penicillin/Streptomycin at 100 IU/ml and 100 μg/ml, respectively)supplemented with 250 μg/ml G418. When performing the luciferase assay,the DMEM basal medium was replaced with phenol-red free DMEM basalmedium and the G418 was omitted. The cells were cultured in an incubatorat 37° C. in 5% CO2 and 95% humidified air.

For NF-κB-mediated luciferase assays, HEK293 NF-κB/luciferase cells wereseeded at 1.5×10⁴ cells/well in 90 μl phenol-red free DMEM completemedium into Corning 3917 white opaque-bottom tissue culture plates. ForFormula II-2, Formula II-4 and Formula II-5A, a 400 μM starting dilutionwas made in 100% DMSO and this dilution was used to generate a 8-pointhalf log dilution series. This dilution series was further diluted 40×in appropriate culture medium and ten μl aliquots were added to the testwells in triplicate resulting in final test concentrations ranging from1 μM to 320 pM. For Formula II-3 and Formula II-5B, a 8 mM startingdilution was made in 100% DMSO and the same procedure was followed asdescribed above resulting in final test concentrations ranging from 20μM to 6.3 nM. The plates were returned to the incubator for 1 hour.After 1 hr pretreatment, 10 μl of a 50 ng/ml TNF-α solution, prepared inthe phenol-red free DMEM medium was added, and the plates were incubatedfor an additional 6 hr. The final concentration of DMSO was 0.25% in allsamples.

At the end of the TNF-α stimulation, 100 μl of Steady Lite HTSluciferase reagent (Packard Bioscience) was added to each well and theplates were left undisturbed for 10 min at room temperature beforemeasuring the luciferase activity. The relative luciferase units (RLU)were measured by using a Fusion microplate fluorometer (PackardBioscience). The EC₅₀ values (the drug concentration at which 50% of themaximal relative luciferase unit inhibition is established) werecalculated in Prism (GraphPad Software) using a sigmoidal dose response,variable slope model.

NF-κB regulates the expression of a large number of genes important ininflammation, apoptosis, tumorigenesis, and autoimmune diseases. Thuscompounds capable of modulating or affecting NF-κB activity are usefulin treating diseases related to inflammation, cancer, and autoimmunediseases, for example. In its inactive form, NF-κB complexes with IκB inthe cytosol and upon stimulation, IκB is phosphorylated, ubiquitinatedand subsequently degraded by the proteasome. The degradation of IκBleads to the activation of NF-κB and its translocation to the nucleus.The effects of Formula II-2, Formula II-3, Formula II-4, Formula II-5Aand Formula II-5B on the activation of NF-κB was evaluated by assessingthe NF-κB-mediated luciferase activity in HEK293 NF-κB/Luc cells uponTNF-α stimulation.

Results from a representative experiment evaluating Formula II-2,Formula II-3 and Formula II-4 (FIG. 44) revealed that pretreatment withFormula II-2 and Formula II-4 resulted in a dose-dependent decrease ofluciferase activity in NF-κB/Luc 293 cells upon TNF-α stimulation. Thecalculated EC₅₀ to inhibit NF-κB inducible luciferase activity in thisexperiment was 73 nM for Formula II-2, while EC₅₀ value for Formula II-4was 67 nM. Similar data were observed in a replicate experiment.

Results from a representative experiment evaluating Formula II-5A andFormula II-5B are shown in FIG. 45 and illustrate that Formula II-5A andFormula II-5B inhibit NF-κB inducible luciferase activity with EC₅₀values of 30 nM and 261 nM respectively. Similar data were observed in areplicate experiment.

Example 44 In vitro Inhibition of Proteasome Activity by Formula II-2,Formula II-3, Formula II-4, Formula II-5A and Formula II-5B

Formula II-2, Formula II-3, Formula II-4, Formula II-5A and FormulaII-5B were prepared as 20 mM stock solution in DMSO and stored in smallaliquots at −80° C. Purified rabbit muscle 20S proteasome was obtainedfrom CalBiochem. To enhance the chymotrypsin-like activity of theproteasome, the assay buffer (20 mM HEPES, pH7.3, 0.5 mM EDTA, and 0.05%Triton X100) was supplemented with SDS resulting in a final SDSconcentration of 0.035%. The substrate used was sucLLVY-AMC, afluorogenic peptide substrate specifically cleaved by thechymotrypsin-like activity of the proteasome. Assays were performed at aproteasome concentration of 1 μg/ml in a final volume of 200 μl in96-well Costar microtiter plates. Formula II-2 and Formula II-4 weretested as eight-point dose response curves with final concentrationsranging from 500 nM to 0.16 nM, while Formula II-3 was tested with finalconcentrations ranging from 10 μM to 3.2 nM. Formula II-5A and FormulaII-5B were tested with final concentrations ranging from 1 μM to 0.32nM. The samples were incubated at 37° C. for five minutes in atemperature controlled plate reader. During the preincubation step, thesubstrate was diluted 25-fold in assay buffer supplemented with 0.035%SDS. After the preincubation period, the reactions were initiated by theaddition of 10 μl of the diluted substrate and the plates were returnedto the plate reader. The final concentration of substrate in thereactions was 20 μM. All data were collected every five minutes for morethan 1.5 hour and plotted as the mean of triplicate data points. TheEC₅₀ values (the drug concentration at which 50% of the maximal relativefluorescence unit is inhibited) were calculated by Prism (GraphPadSoftware) using a sigmoidal dose-response, variable slope model.

Results from a representative experiment evaluating Formula II-2,Formula II-3 and Formula II-4 are shown in FIG. 46 and illustrate thatFormula II-2 and Formula II-4 inhibit the chymotrypsin-like activity ofthe proteasome with EC₅₀ values of 18.5 nM and 15 nM respectively.Formula II-3 is active in this assay with an EC₅₀ value of 890 nM.Similar results were obtained from an independent experiment.

Results from a representative experiment evaluating Formula II-5A andFormula II-5B are shown in FIG. 47 and illustrate that Formula II-5A andFormula II-5B inhibit the chymotrypsin-like activity of the proteasomewith EC₅₀ values of 6 nM and 88 nM respectively. Similar results wereobtained in an independent experiment.

Example 45 Inhibition of Anthrax Lethal Toxin

Anthrax toxin is responsible for the symptoms associated with anthrax.In this disease, B. anthracis spores are inhaled and lodge in the lungswhere they are ingested by macrophages. Within the macrophage, sporesgerminate, replicate, resulting in killing of the cell. Before killingoccurs, however, infected macrophages migrate to the lymph nodes where,upon death, they release their contents, allowing the organism to enterthe bloodsteam, further replicate, and secrete lethal toxins.

Two proteins called protective antigen (PA 83 kDa) and lethal factor(LF, 90 kDa), play a key role in the pathogenesis of anthrax. Theseproteins are collectively known as lethal toxin (LeTx). When combined,PA and LF cause death when injected intravenously in animals. Lethaltoxin is also active in a few cell culture lines of macrophages causingcell death within a few hours. LeTx can induce both necrosis andapoptosis in mouse macrophage-like RAW264.7 cells upon in vitrotreatment.

In vitro Cell-Based Assay for Inhibitors of Lethal Toxin-MediatedCytotoxicity

RAW264.7 cells (obtained from the American Type Culture Collection) wereadapted to and maintained in RPMI-1640 medium supplemented with 10%fetal bovine serum, 2 mM L-glutamine and 1% Penicillin/Streptomycin(complete medium) at 37° C. in a humidified 5% CO₂ incubator. For theassay, cells were plated overnight in complete medium at a concentrationof 50,000 cells/well in a 96-well plate. Media was removed the followingday and replaced with serum-free complete medium with or without varyingconcentrations of Formulae II-2, II-3, II-4, II-5A, II-5B, II-13C,II-17, II-18 and IV-3C starting at 330 nM and diluting at ½ logintervals for an 8-point dose-response. After a 45 minute preincubation,1 μg/ml LF and 1 μg/ml PA alone or in combination (LF:PA, also termedlethal toxin (LeTx)) were added to cells. Recombinant LF and PA wereobtained from List Biological Laboratories. Additional plates with noLeTx added were included as a control. Cells were then incubated for sixhours followed by addition of 0.02 mg/ml resazurin dye (MolecularProbes, Eugene, Oreg.) prepared in Mg++, Ca++ free PBS (Mediatech,Herndon, Va.). Plates were then incubated an additional 1.5 hours priorto the assessment of cell viability. Since resazurin is metabolized byliving cells, cytotoxicity or cell viability can be assessed bymeasuring fluorescence using 530 excitation and 590 emission filters.Data are expressed as the percent viability as compared to a DMSO alonecontrol (high) and the LeTx alone control (low) using the followingequation: Percent viability=100*((Measured OD-low control)/(highcontrol-low control)).

Inhibition of Anthrax Lethal Toxin-mediated Cytotoxicity in RAW 264.7Cells

Data in FIG. 48 summarize the effects of Formula II-2, Formula II-3 andFormula II-4 against LeTx-mediated cytotoxicity of the RAW 264.7 murinemacrophage-like cell line. Treatment of RAW 264.7 cells with FormulaII-2 and Formula II-4 resulted in an increase in the viability of LeTxtreated cells with EC₅₀ values of 14 nM (FIG. 48). The EC₅₀ values forFormula II-3 for LeTx protection was not be determined at theconcentrations tested (EC₅₀>330 nM, the maximum concentrationevaluated). Data in Table 18 show the effects of Formulae II-5A, II-5B,II-13C, II-17, II-18 and IV-3C against LeTx-mediated cytotoxicity of theRAW 264.7 murine macrophage-like cell line. Treatment of RAW 264.7 cellswith Formula II-5A and II-18 showed an increase in the viability of LeTxtreated RAW 264.7 cells with EC₅₀ values of 3 nM and 4 nM respectively.Treatment with Formula II-17 and Formula II-5B resulted in an increasein the viability of LeTx treated cells with EC₅₀ values of 42 nM and 45nM respectively. The EC₅₀ values for Formulae II-13C and IV-3C for LeTxprotection could not be determined at the concentrations tested(EC₅₀>330 nM, the maximum concentration evaluated). TABLE 18 EC₅₀ valuesfor inhibition of RAW 264.7 cell cytotoxicity mediated by anthrax lethaltoxin Compound EC₅₀ (nM) Formula II-17  42 Formula II-18   4 FormulaII-5A   3 Formula II-5B  45 Formula II-13C >330 nM Formula IV-3C >330 nM

Example 46 Formulation to be Administered Orally or the Like

A mixture obtained by thoroughly blending 1 g of a compound obtained andpurified by the method of the embodiment, 98 g of lactose and 1 g ofhydroxypropyl cellulose is formed into granules by any conventionalmethod. The granules are thoroughly dried and sifted to obtain a granulepreparation suitable for packaging in bottles or by heat sealing. Theresultant granule preparations are orally administered at betweenapproximately 100 ml/day to approximately 1000 ml/day, depending on thesymptoms, as deemed appropriate by those of ordinary skill in the art oftreating cancerous tumors in humans.

The examples described above are set forth solely to assist in theunderstanding of the embodiments. Thus, those skilled in the art willappreciate that the methods may provide derivatives of compounds.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand procedures described herein are presently representative ofpreferred embodiments and are exemplary and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the embodiments disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions indicates the exclusion of equivalents of the features shownand described or portions thereof. It is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be falling within the scope of theinvention, which is limited only by the following claims.

1. A method of treating a neoplastic disease in an animal, the methodcomprising: administering to the animal, a therapeutically effectiveamount of a compound of a formula selected from Formulae I-V, andpharmaceutically acceptable salts and pro-drug esters thereof:
 2. Themethod of claim 1, wherein the neoplastic disease is cancer.
 3. Themethod of claim 2, wherein the cancer is selected from the groupconsisting of breast cancer, sarcoma, leukemia, ovarian cancer, uretalcancer, bladder cancer, prostate cancer, colon cancer, rectal cancer,stomach cancer, lung cancer, lymphoma, multiple myeloma, pancreaticcancer, liver cancer, kidney cancer, endocrine cancer, skin cancer,melanoma, angioma, and brain or central nervous system (CNS) cancer. 4.The method of claim 3, wherein the cancer is a multiple myeloma, acolorectal carcinoma, a prostate carcinoma, a breast adenocarcinoma, anon-small cell lung carcinoma, an ovarian carcinoma or a melanoma. 5.The method of claim 2, wherein the cancer is a drug resistant cancer. 6.The method of claim 5, wherein the drug-resistant cancer displays atleast one of the following: elevated levels of the P-glycoprotein effluxpump, increased expression of the multidrug-resistance associatedprotein 1 encoded by MRP1, reduced drug uptake, alteration of the drug'starget or increasing repair of drug-induced DNA damage, alteration ofthe apoptotic pathway or the activation of cytochrome P450 enzymes. 7.The method of claim 5, wherein the drug resistant cancer is a sarcoma ora leukemia.
 8. The method of claim 1, wherein the animal is a mammal. 9.The method of claim 1, wherein the animal is a human.
 10. The method ofclaim 1, wherein the animal is a rodent.
 11. The method of claim 1,wherein the the compound is:

wherein R₈ is selected from the group consisting of H, F, Cl, Br and I.12. The method of claim 1, wherein the compound is:

wherein R₈ is selected from the group consisting of H, F, Cl, Br, and I.13. The method of claim 1, further comprising the steps of: identifyinga subject that would benefit from administration of an anticancer agent;performing the method on the subject.
 14. A pharmaceutical compositioncomprising a compound of a formula selected from Formulae I-V, andpharmaceutically acceptable salts and pro-drug esters thereof.
 15. Thepharmaceutical composition of claim 14, further comprising ananti-microbial agent.
 16. A method of inhibiting the growth of a cancercell, comprising contacting a cancer cell with a compound of a formulaselected from Formulae I-V, and pharmaceutically acceptable salts andpro-drug esters thereof.
 17. The method of claim 16, wherein the cancercell is a multiple myeloma, a colorectal carcinoma, a prostatecarcinoma, a breast adenocarcinoma, a non-small cell lung carcinoma, anovarian carcinoma and a melanoma.
 18. A method of inhibiting proteasomeactivity comprising the step contacting a cell with a compound of aformula selected from Formulae I-V, and pharmaceutically acceptablesalts and pro-drug esters thereof.
 19. A method of inhibiting NF-κBactivation comprising the step contacting a cell with a compound of aformula selected from Formulae I-V, and pharmaceutically acceptablesalts and pro-drug esters thereof.
 20. A method for treating aninflammatory condition, comprising administering an effective amount ofa compound of a formula selected from Formulae I-V to a patient in needthereof.
 21. The method of claim 20, wherein the inflammatory conditionis selected from the group consisting of rheumatoid arthritis, asthma,multiple sclerosis, psoriasis, stroke, and myocardial infarction.
 22. Amethod for treating a microbial illness comprising administering aneffective amount of a compound of a formula selected from Formulae I-Vto a patient in need thereof.
 23. The method of claim 22, wherein themicrobial illness is caused by a microbe selected from the groupconsisting of B. anthracis, Plasmodium, Leishmania, and Trypanosoma.